Nucleic acid molecules encoding polypeptides involved in regulation of sugar and lipid metabolism and methods of use VIII

ABSTRACT

This invention relates generally to nucleic acid sequences encoding proteins that are related to the presence of seed storage compounds in plants. More specifically, the present invention relates to  Arabidopsis thaliana, Brassica napus, Glycine max  and  Oryza sativa  nucleic acid sequences encoding sugar and lipid metabolism regulator proteins and the use of these sequences in transgenic plants. In particular, the invention is directed to methods for manipulating sugar-related compounds and for increasing oil level and altering the fatty acid composition in plants and seeds. The invention further relates to methods of using these novel plant polypeptides to stimulate plant growth and/or to increase yield and/or composition of seed storage compounds.

RELATED APPLICATIONS

This application is a national stage application (under 35 U.S.C. §371)of PCT/EP2006/069271, filed Dec. 4, 2006, which claims benefit of U.S.provisional application 60/597,558, filed Dec. 9, 2005.

SEQUENCE LISTING SUBMISSION

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety. The name of the text file containingthe Sequence Listing is Revised Sequence List 13156 00170 US. The sizeof the text file is 3,378 KB, and the text file was created on Aug. 27,2008.

Described herein are inventions in the field of genetic engineering ofplants, including isolated nucleic acid molecules to improve agronomic,horticultural, and quality traits. This invention relates generally tonucleic acid sequences encoding proteins that are related to thepresence of seed storage compounds in plants. More specifically, thepresent invention relates to Arabidopsis thaliana, Brassica napus,Glycine max and Oryza sativa nucleic acid sequences encoding sugar andlipid metabolism regulator proteins and the use of these sequences intransgenic plants. In particular, the invention is directed to methodsfor manipulating sugar-related compounds and for increasing oil leveland altering the fatty acid composition in plants and seeds. Theinvention further relates to methods of using these novel plantpolypeptides to stimulate plant growth and/or to increase yield and/orcomposition of seed storage compounds.

The study and genetic manipulation of plants has a long history thatbegan even before the framed studies of Gregor Mendel. In perfectingthis science, scientists have accomplished modification of particulartraits in plants ranging from potato tubers having increased starchcontent to oilseed plants such as canola and sunflower having increasedor altered fatty acid content. With the increased consumption and use ofplant oils, the modification of seed oil content and seed oil levels hasbecome increasingly widespread (e.g. Töpfer et al. 1995, Science268:681-686). Manipulation of biosynthetic pathways in transgenic plantsprovides a number of opportunities for molecular biologists and plantbiochemists to affect plant metabolism giving rise to the production ofspecific higher-value products. The seed oil production or compositionhas been altered in numerous traditional oilseed plants such as soybean(U.S. Pat. No. 5,955,650), canola (U.S. Pat. No. 5,955,650), sunflower(U.S. Pat. No. 6,084,164), and rapeseed (Töpfer et al. 1995, Science268:681-686), and non-traditional oil seed plants such as tobacco(Cahoon et al. 1992, Proc. Natl. Acad. Sci. USA 89:11184-11188).

Plant seed oils comprise both neutral and polar lipids (see Table 13).The neutral lipids contain primarily triacylglycerol, which is the mainstorage lipid that accumulates in oil bodies in seeds. The polar lipidsare mainly found in the various membranes of the seed cells, e.g. theendoplasmic reticulum, microsomal membranes, and the cell membrane. Theneutral and polar lipids contain several common fatty acids (see Table14) and a range of less common fatty acids. The fatty acid compositionof membrane lipids is highly regulated and only a select number of fattyacids are found in membrane lipids. On the other hand, a large number ofunusual fatty acids can be incorporated into the neutral storage lipidsin seeds of many plant species (Van de Loo F. J. et al. 1993, “UnusualFatty Acids in Lipid Metabolism in Plants,” pp. 91-126, editor T S MooreJr., CRC Press; Millar et al., 2000, Trends Plant Sci., 5:95-101).

Lipids are synthesized from fatty acids, and their synthesis may bedivided into two parts: The prokaryotic pathway and the eukaryoticpathway (Browse et al. 1986, Biochemical J. 235:25-31; Ohlrogge & Browse1995, Plant Cell 7:957-970). The prokaryotic pathway is located inplastids that are the primary site of fatty acid biosynthesis. Fattyacid synthesis begins with the conversion of acetyl-CoA to malonyl-CoAby acetyl-CoA carboxylase (ACCase). Malonyl-CoA is converted tomalonyl-acyl carrier protein (ACP) by the malonyl-CoA:ACP transacylase.The enzyme beta-keto-acyl-ACP-synthase III (KAS III) catalyzes acondensation reaction, in which the acyl group from acetyl-CoA istransferred to malonyl-ACP to form 3-ketobutyryl-ACP. In a subsequentseries of condensation, reduction, and dehydration reactions, thenascent fatty acid chain on the ACP cofactor is elongated by thestep-by-step addition (condensation) of two carbon atoms donated bymalonyl-ACP until a 16- or 18-carbon saturated fatty acid chain isformed. The plastidial delta-9 acyl-ACP desaturase introduces the firstunsaturated double bond into the fatty acid. Thioesterases cleave thefatty acids from the ACP cofactor and free fatty acids are exported tothe cytoplasm, where they participate as fatty acyl-CoA esters in theeukaryotic pathway. In this pathway the fatty acids are esterified byglycerol-3-phosphate acyltransferase and lysophosphatidic acidacyl-transferase to the sn-1 and sn-2 positions of glycerol-3-phosphate,respectively, to yield phosphatidic acid (PA). The PA is the precursorfor other polar and neutral lipids, the latter being formed in theKennedy pathway (Voelker 1996, Genetic Engineering ed.: Setlow18:111-113; Shanklin & Cahoon 1998, Annu. Rev. Plant Physiol. Plant Mol.Biol. 49:611-641; Frentzen 1998, Lipids 100:161-166; Millar et al. 2000,Trends Plant Sci. 5:95-101).

Storage lipids in seeds are synthesized from carbohydrate-derivedprecursors. Plants have a complete glycolytic pathway in the cytosol(Plaxton 1996, Annu. Rev. Plant Physiol. Plant Mol. Biol. 47:185-214),and it has been shown that a complete pathway also exists in theplastids of rapeseeds (Kang & Rawsthorne 1994, Plant J. 6:795-805).Sucrose is the primary source of carbon and energy, transported from theleaves into the developing seeds. During the storage phase of seeds,sucrose is converted in the cytosol to provide the metabolic precursorsglucose-6-phosphate and pyruvate. These are transported into theplastids and converted into acetyl-CoA that serves as the primaryprecursor for the synthesis of fatty acids. Acetyl-CoA in the plastidsis the central precursor for lipid biosynthesis. Acetyl-CoA can beformed in the plastids by different reactions and the exact contributionof each reaction is still being debated (Ohlrogge & Browse 1995, PlantCell 7:957-970). It is however accepted that a large part of theacetyl-CoA is derived from glucose-6-phosphate and pyruvate that areimported from the cytoplasm into the plastids. Sucrose is produced inthe source organs (leaves, or anywhere that photosynthesis occurs) andis transported to the developing seeds that are also termed sink organs.In the developing seeds, sucrose is the precursor for all the storagecompounds, i.e. starch, lipids, and partly the seed-storage proteins.Therefore, it is clear that carbohydrate metabolism, in which sucroseplays a central role, is very important to the accumulation of seedstorage compounds.

Storage compounds, such as triacylglycerols (seed oil), serve as carbonand energy reserves, which are used during germination and growth of theyoung seedling. Seed (vegetable) oil is also an essential component ofthe human diet and a valuable commodity providing feed stocks for thechemical industry.

Although the lipid and fatty acid content, and/or composition of seedoil, can be modified by the traditional methods of plant breeding, theadvent of recombinant DNA technology has allowed for easier manipulationof the seed oil content of a plant, and, in some cases, has allowed forthe alteration of seed oils in ways that could not be accomplished bybreeding alone (see, e.g., Töpfer et al. 1995, Science 268:681-686). Forexample, introduction of a Δ¹²-hydroxylase nucleic acid sequence intotransgenic tobacco resulted in the introduction of a novel fatty acid,ricinoleic acid, into the tobacco seed oil (Van de Loo et al. 1995,Proc. Natl. Acad. Sci. USA 92:6743-6747). Tobacco plants have also beenengineered to produce low levels of petroselinic acid by theintroduction and expression of an acyl-ACP desaturase from coriander(Cahoon et al. 1992, Proc. Natl. Acad. Sci. USA 89:11184-11188).

The modification of seed oil content in plants has significant medical,nutritional, and economic ramifications. With regard to the medicalramifications, the long chain fatty acids (C18 and longer) found in manyseed oils have been linked to reductions in hypercholesterolemia andother clinical disorders related to coronary heart disease (Brenner1976, Adv. Exp. Med. Biol. 83:85-101). Therefore, consumption of a planthaving increased levels of these types of fatty acids may reduce therisk of heart disease. Enhanced levels of seed oil content also increaselarge-scale production of seed oils and thereby reduce the cost of theseoils.

In order to increase or alter the levels of compounds such as seed oilsin plants, nucleic acid sequences and proteins regulating lipid andfatty-acid metabolism must be identified. As mentioned earlier, severaldesaturase nucleic acids such as the Δ⁶-desaturase nucleic acid,Δ¹²-desaturase nucleic acid, and acyl-ACP desaturase nucleic acid havebeen cloned and demonstrated to encode enzymes required for fatty acidsynthesis in various plant species. Oleosin nucleic acid sequences fromsuch different species as canola, soybean, carrot, pine, and Arabidopsisthaliana have also been cloned and determined to encode proteinsassociated with the phospholipid monolayer membrane of oil bodies inthose plants.

It has also been determined that two phytohormones, gibberellic acid(GA), and absisic acid (ABA), are involved in overall regulatoryprocesses in seed development (e.g. Ritchie & Gilroy 1998, PlantPhysiol. 116:765-776; Arenas-Huertero et al. 2000, Genes Dev.14:2085-2096). Both the GA and ABA pathways are affected by okadaicacid, a protein phosphatase inhibitor (Kuo et al. 1996, Plant Cell.8:259-269). The regulation of protein phosphorylation by kinases andphosphatases is accepted as a universal mechanism of cellular control(Cohen 1992, Trends Biochem. Sci. 17:408-413). Likewise, the planthormones ethylene (e.g. Zhou et al. 1998, Proc. Natl. Acad. Sci. USA95:10294-10299; Beaudoin et al. 2000, Plant Cell 2000:1103-1115), andauxin (e.g. Colon-Carmona et al. 2000, Plant Physiol. 124:1728-1738) areinvolved in controlling plant development as well.

Thus, the technical problem underlying the present invention may be seenas the provision of means and methods for complying with theaforementioned needs. The technical problem is solved by the embodimentscharacterized in the claims and herein below. In principle, thisinvention discloses nucleic acid sequences from Arabidopsis thaliana,Brassica napus, Glycine max and Oryza sativa. These nucleic acidsequences can be used to alter or increase the levels of seed storagecompounds such as proteins, sugars, and oils in plants, includingtransgenic plants, such as canola, linseed, soybean, sunflower, maize,oat, rye, barley, wheat, rice, pepper, tagetes, cotton, oil palm,coconut palm, flax, castor, and peanut, which are oilseed plantscontaining high amounts of lipid compounds.

Accordingly, the present invention relates to a polynucleotide whichcomprises a nucleic acid sequence selected from the group consisting of:

-   (a) a nucleic acid sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9,    11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,    45, 47, 63, 65, 67, 69, 71, 73, 75, 77, 87, 89, 91, 93, 95, 97, 99,    101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,    127, 129, 131, 133, 135, 137, 158, 160, 162, 164, 166, 168, 170,    172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196,    198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222,    224, 226, 228, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256,    258, 260, 262, 264, 266, 273, 323, 325, 327, 329, 331, 333, 335,    337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361,    363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387,    389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413,    415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439,    441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465,    467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491,    493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517,    519, 521, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572,    574, 576, 578, 580, 582, 591, 593, 595, 597, 599, 601, 603, 605,    607, 609, 611, 613, 615, 625, 627, 629, 631, 633, 635, 657, 659,    661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685,    687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711,    713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737,    739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763,    765, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800,    802, 804, or 806;-   (b) a nucleic acid sequence encoding a polypeptide having an amino    acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,    20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 64, 66,    68, 70, 72, 74, 76, 78, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106,    108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132,    134, 136, 138, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177,    179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203,    205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,    241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265,    267, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296,    298, 300, 302, 304, 306, 308, 310, 312, 314, 322, 324, 326, 328,    330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354,    356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380,    382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406,    408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432,    434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458,    460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484,    486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510,    512, 514, 516, 518, 520, 522, 553, 555, 557, 559, 561, 563, 565,    567, 569, 571, 573, 575, 577, 579, 581, 583, 592, 594, 596, 598,    600, 602, 604, 606, 608, 610, 612, 614, 616, 626, 628, 630, 632,    634, 636, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678,    680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704,    706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730,    732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756,    758, 760, 762, 764, 766, 779, 781, 783, 785, 787, 789, 791, 793,    795, 797, 799, 801, 803, 805 or 807;-   (c) a nucleic acid sequence which is at least 70% identical to the    nucleic acid sequence of (a) or (b), wherein said nucleic acid    sequence encodes a polypeptide or biologically active portion    thereof being capable of increasing the seed storage compound    content when expressed in transgenic plants; and-   (d) a nucleic acid sequence being a fragment of any one of (a) to    (c), wherein said fragment encodes a polypeptide or biologically    active portion thereof being capable of increasing the seed storage    compound content when expressed in transgenic plants.

The term “polynucleotide” as used in accordance with the presentinvention relates to a polynucleotide comprising a nucleic acid sequencewhich encodes a polypeptide being capable of increasing the seed storagecompound content and, preferably, the total fatty acid content whenexpressed in transgenic plants. More preferably, the polypeptide encodedby the polynucleotide of the present invention has a biological activityas indicated in Table 15. The polypeptides encoded by the polynucleotideof the present invention are also referred to as lipid metabolismproteins (LMP) herein below. Suitable assays for measuring theactivities mentioned before are well known in the art and are describedin the accompanying Examples. Preferably, the polynucleotide of thepresent invention upon expression in a plant seed, preferably in a plantseed of the Arabidopsis thaliana ecotype Columbia-2, shall be capable ofsignificantly increasing the total fatty acid content. How to determinewhether an increase is significant is described elsewhere in thisspecification. Further details are to be found in the accompanyingExamples, below.

Preferably, the polynucleotide of the present invention upon expressionin the seed of a transgenic plant is capable of significantly increasingthe amount by weight of at least one seed storage compound, morepreferably, of the fatty acids. More preferably, such an increase asreferred to in accordance with the present invention is an increase ofthe amount by weight of at least 1, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20,22.5 or 25% as compared to a control. Whether an increase is significantcan be determined by statistical tests well known in the art including,e.g., Student's t-test. The percent increase rates of a seed storagecompound are, preferably, determined compared to an empty vectorcontrol. An empty vector control is a transgenic plant, which has beentransformed with the same vector or construct as a transgenic plantaccording to the present invention except for such a vector or constructis lacking the polynucleotide of the present invention. Alternatively,an untreated plant (i.e. a plant which has not been geneticallymanipulated) may be used as a control.

A polynucleotide encoding a polypeptide having a biological activity asspecified above has been obtained in accordance with the presentinvention from Arabidopsis thaliana, Brassica napus or Glycine max. Thecorresponding polynucleotides, preferably, comprises the nucleic acidsequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 63, 65, 67, 69, 71, 73,75, 77, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113,115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 158, 160,162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188,190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216,218, 220, 222, 224, 226, 228, 238, 240, 242, 244, 246, 248, 250, 252,254, 256, 258, 260, 262, 264, 266, 273, 323, 325, 327, 329, 331, 333,335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361,363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389,391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417,419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445,447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473,475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501,503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 552, 554, 556, 558,560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 591, 593,595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 625, 627, 629,631, 633, 635, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677,679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705,707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733,735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761,763, 765, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800,802, 804, or 806 encoding a polypeptide having the amino acid sequenceof SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 64, 66, 68, 70, 72, 74, 76, 78, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 159, 161, 163, 165,167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193,195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221,223, 225, 227, 229, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259,261, 263, 265, 267, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292,294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 322, 324, 326,328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354,356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382,384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410,412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438,440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466,468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494,496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522,553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579,581, 583, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614,616, 626, 628, 630, 632, 634, 636, 658, 660, 662, 664, 666, 668, 670,672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698,700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726,728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754,756, 758, 760, 762, 764, 766, 779, 781, 783, 785, 787, 789, 791, 793,795, 797, 799, 801, 803, 805 or 807.

It is to be understood that a polypeptide having the aforementionedspecific amino acid sequences may be encoded due to the degeneratedgenetic code by other polynucleotides as well.

Moreover, the term “polynucleotide” as used in accordance with thepresent invention further encompasses variants of the aforementionedspecific polynucleotides. Said variants may represent orthologs,paralogs or other homologs of the polynucleotide of the presentinvention.

The polynucleotide variants, preferably, also comprise a nucleic acidsequence characterized in that the sequence can be derived from theaforementioned specific nucleic acid sequences shown in SEQ ID NO: 1, 3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 63, 65, 67, 69, 71, 73, 75, 77, 87, 89, 91, 93, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 131, 133, 135, 137, 158, 160, 162, 164, 166, 168, 170, 172, 174,176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202,204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 238,240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266,273, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347,349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375,377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403,405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431,433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459,461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487,489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515,517, 519, 521, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572,574, 576, 578, 580, 582, 591, 593, 595, 597, 599, 601, 603, 605, 607,609, 611, 613, 615, 625, 627, 629, 631, 633, 635, 657, 659, 661, 663,665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691,693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719,721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747,749, 751, 753, 755, 757, 759, 761, 763, 765, 778, 780, 782, 784, 786,788, 790, 792, 794, 796, 798, 800, 802, 804, or 806 by at least onenucleotide substitution, addition and/or deletion whereby the variantnucleic acid sequence shall still encode a polypeptide having abiological activity as specified above. Variants also encompasspolynucleotides comprising a nucleic acid sequence which is capable ofhybridizing to the aforementioned specific nucleic acid sequences,preferably, under stringent hybridization conditions. These stringentconditions are known to the skilled worker and can be found in CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. A preferred example for stringent hybridization conditionsare hybridization conditions in 6× sodium chloride/sodium citrate (=SSC)at approximately 45° C., followed by one or more wash steps in 0.2×SSC,0.1% SDS at 50 to 65° C. The skilled worker knows that thesehybridization conditions differ depending on the type of nucleic acidand, for example when organic solvents are present, with regard to thetemperature and concentration of the buffer. For example, under“standard hybridization conditions” the temperature differs depending onthe type of nucleic acid between 42° C. and 58° C. in aqueous bufferwith a concentration of 0.1 to 5×SSC (pH 7.2). If organic solvent ispresent in the abovementioned buffer, for example 50% formamide, thetemperature under standard conditions is approximately 42° C. Thehybridization conditions for DNA:DNA hybrids are, preferably, 0.1×SSCand 20° C. to 45° C., preferably between 30° C. and 45° C. Thehybridization conditions for DNA:RNA hybrids are, preferably, 0.1×SSCand 30° C. to 55° C., preferably between 45° C. and 55° C. Theabovementioned hybridization temperatures are determined for example fora nucleic acid with approximately 100 bp (=base pairs) in length and aG+C content of 50% in the absence of formamide. The skilled worker knowshow to determine the hybridization conditions required by referring totextbooks such as the textbook mentioned above, or the followingtextbooks: Sambrook et al., “Molecular Cloning”, Cold Spring HarborLaboratory, 1989; Hames and Higgins (Ed.) 1985, “Nucleic AcidsHybridization: A Practical Approach”, IRL Press at Oxford UniversityPress, Oxford; Brown (Ed.) 1991, “Essential Molecular Biology: APractical Approach”, IRL Press at Oxford University Press, Oxford.Alternatively, polynucleotide variants are obtainable by PCR-basedtechniques such as mixed oligonucleotide primer-based amplification ofDNA, i.e. using degenerated primers against conserved domains of thepolypeptides of the present invention. Conserved domains of thepolypeptide of the present invention may be identified by a sequencecomparison of the nucleic acid sequences of the polynucleotides or theamino acid sequences of the polypeptides of the present invention.Oligonucleotides suitable as PCR primers as well as suitable PCRconditions are described in the accompanying Examples. As a template,DNA or cDNA from bacteria, fungi, plants or animals may be used.Further, variants include polynucleotides comprising nucleic acidsequences which are at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 98% or at least 99% identicalto the nucleic acid sequences shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 63,65, 67, 69, 71, 73, 75, 77, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105,107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133,135, 137, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 238, 240, 242, 244,246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 273, 323, 325,327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353,355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381,383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409,411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437,439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465,467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493,495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521,552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578,580, 582, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613,615, 625, 627, 629, 631, 633, 635, 657, 659, 661, 663, 665, 667, 669,671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697,699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725,727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753,755, 757, 759, 761, 763, 765, 778, 780, 782, 784, 786, 788, 790, 792,794, 796, 798, 800, 802, 804, or 806 retaining a biological activity asspecified above. Moreover, also encompassed are polynucleotides whichcomprise nucleic acid sequences encoding amino acid sequences which areat least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98% or at least 99% identical to the amino acidsequences shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 64, 66, 68, 70, 72,74, 76, 78, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 159,161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187,189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215,217, 219, 221, 223, 225, 227, 229, 241, 243, 245, 247, 249, 251, 253,255, 257, 259, 261, 263, 265, 267, 274, 276, 278, 280, 282, 284, 286,288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314,322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348,350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376,378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404,406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432,434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460,462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488,490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516,518, 520, 522, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573,575, 577, 579, 581, 583, 592, 594, 596, 598, 600, 602, 604, 606, 608,610, 612, 614, 616, 626, 628, 630, 632, 634, 636, 658, 660, 662, 664,666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692,694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720,722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748,750, 752, 754, 756, 758, 760, 762, 764, 766, 779, 781, 783, 785, 787,789, 791, 793, 795, 797, 799, 801, 803, 805 or 807 wherein thepolypeptide comprising the amino acid sequence retains a biologicalactivity as specified above. The percent identity values are,preferably, calculated over the entire amino acid or nucleic acidsequence region. A series of programs based on a variety of algorithmsis available to the skilled worker for comparing different sequences. Inthis context, the algorithms of Needleman and Wunsch or Smith andWaterman give particularly reliable results. To carry out the sequencealignments, the program PileUp (J. Mol. Evolution., 25, 351-360, 1987,Higgins et al., CABIOS, 5 1989: 151-153) or the programs Gap and BestFit(Needleman and Wunsch (J. Mol. Biol. 48; 443-453 (1970)) and Smith andWaterman (Adv. Appl. Math. 2; 482-489 (1981))), which are part of theGCG software packet [Genetics Computer Group, 575 Science Drive,Madison, Wis., USA 53711 (1991)], are to be used. The sequence identityvalues recited above in percent (%) are to be determined, preferably,using the program GAP over the entire sequence region with the followingsettings: Gap Weight: 50, Length Weight: 3, Average Match: 10.000 andAverage Mismatch: 0.000, which, unless otherwise specified, shall alwaysbe used as standard settings for sequence alignments. For the purposesof the invention, the percent sequence identity between two nucleic acidor polypeptide sequences can be also determined using the Vector NTI 7.0(PC) software package (InforMax, 7600 Wisconsin Ave., Bethesda, Md.20814). A gap-opening penalty of 15 and a gap extension penalty of 6.66are used for determining the percent identity of two nucleic acids. Agap-opening penalty of 10 and a gap extension penalty of 0.1 are usedfor determining the percent identity of two polypeptides. All otherparameters are set at the default settings. For purposes of a multiplealignment (Clustal W algorithm), the gap-opening penalty is 10, and thegap extension penalty is 0.05 with blosum62 matrix. It is to beunderstood that for the purposes of determining sequence identity whencomparing a DNA sequence to an RNA sequence, a thymidine nucleotidesequence is equivalent to a uracil nucleotide.

A polynucleotide comprising a fragment of any of the aforementionednucleic acid sequences is also encompassed as a polynucleotide of thepresent invention. The fragment shall encode a polypeptide which stillhas a biological activity as specified above. Accordingly, thepolypeptide may comprise or consist of the domains of the polypeptide ofthe present invention conferring the said biological activity. Afragment as meant herein, preferably, comprises at least 20, at least50, at least 100, at least 250 or at least 500 consecutive nucleotidesof any one of the aforementioned nucleic acid sequences or encodes anamino acid sequence comprising at least 20, at least 30, at least 50, atleast 80, at least 100 or at least 150 consecutive amino acids of anyone of the aforementioned amino acid sequences.

More preferably, said variant polynucleotides encoding a variantpolypeptide of the polypeptide shown in SEQ ID NO: 2 comprise at leastone of the amino acid sequence patterns shown in any one of SEQ ID NOs:49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 or 62. Morepreferably, said variant polynucleotides encoding a variant polypeptideof the polypeptide shown in SEQ ID NO: 64 comprise at least one of theamino acid sequence patterns shown in any one of SEQ ID NOs: 79, 80, 81,82, 83, 84, 85 or 86. More preferably, said variant polynucleotidesencoding a variant polypeptide of the polypeptide shown in SEQ ID NO: 88comprise at least one of the amino acid sequence patterns shown in anyone of SEQ ID NOs: 149, 150, 151, 152, 153, 154, 155, 156 or 157. Morepreferably, said variant polynucleotides encoding a variant polypeptideof the polypeptide shown in SEQ ID NO: 159 comprise at least one of theamino acid sequence patterns shown in any one of SEQ ID NOs: 230, 231,232, 233, 234, 235, 236 or 237. More preferably, said variantpolynucleotides encoding a variant polypeptide of the polypeptide shownin SEQ ID NO: 239 comprise at least one of the amino acid sequencepatterns shown in any one of SEQ ID NOs: 268 269, 270, 271 or 272. Morepreferably, said variant polynucleotides encoding a variant polypeptideof the polypeptide shown in SEQ ID NO: 274 comprise at least one of theamino acid sequence patterns shown in any one of SEQ ID NOs: 315, 316,317, 318, 319 or 320. More preferably, said variant polynucleotidesencoding a variant polypeptide of the polypeptide shown in SEQ ID NO:322 comprise at least one of the amino acid sequence patterns shown inany one of SEQ ID NOs: 541, 542, 543, 544, 545, 546, 547, 548, 549, 550or 551. More preferably, said variant polynucleotides encoding a variantpolypeptide of the polypeptide shown in SEQ ID NO: 553 comprise at leastone of the amino acid sequence patterns shown in any one of SEQ ID NOs:586, 587, 588, 589, 590, 549, 550 or 551. More preferably, said variantpolynucleotides encoding a variant polypeptide of the polypeptide shownin SEQ ID NO: 592 comprise at least one of the amino acid sequencepatterns shown in any one of SEQ ID NOs: 621, 622, 623, 624, 549, 550 or551. More preferably, said variant polynucleotides encoding a variantpolypeptide of the polypeptide shown in SEQ ID NO: 626 comprise at leastone of the amino acid sequence patterns shown in any one of SEQ ID NOs:643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655 or 656.More preferably, said variant polynucleotides encoding a variantpolypeptide of the polypeptide shown in SEQ ID NO: 658 comprise at leastone of the amino acid sequence patterns shown in any one of SEQ ID NOs:771, 772, 773, 774, 775, 776 or 777. More preferably, said variantpolynucleotides encoding a variant polypeptide of the polypeptide shownin SEQ ID NO: 779 comprise at least one of the amino acid sequencepatterns shown in any one of SEQ ID NOs: 816, 817, 818, 819, 820, 821,822, 823 or 824.

Furthermore preferably, a variant polynucleotide of a polynucleotideencoding an amino acid sequence as shown in SEQ ID NO: 2 encodes anamino acid sequence as shown in any one of SEQ ID NOs.: 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 or48, a variant polynucleotide of a polynucleotide encoding an amino acidsequence as shown in SEQ ID NO: 64 encodes an amino acid sequence asshown in any one of SEQ ID NOs.: 66, 68, 70, 72, 74, 76 or 78, a variantpolynucleotide of a polynucleotide encoding an amino acid sequence asshown in SEQ ID NO: 88 encodes an amino acid sequence as shown in anyone of SEQ ID NOs.: 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110,112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136 or 138,a variant polynucleotide of a polynucleotide encoding an amino acidsequence as shown in SEQ ID NO: 159 encodes an amino acid sequence asshown in any one of SEQ ID NOs.: 161, 163, 165, 167, 169, 171, 173, 175,177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203,205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227 or 229, avariant polynucleotide of a polynucleotide encoding an amino acidsequence as shown in SEQ ID NO: 239 encodes an amino acid sequence asshown in any one of SEQ ID NOs.: 241, 243, 245, 247, 249, 251, 253, 255,257, 259, 261, 263, 265 or 267, a variant polynucleotide of apolynucleotide encoding an amino acid sequence as shown in SEQ ID NO:274 encodes an amino acid sequence as shown in any one of SEQ ID NOs.:276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302,304, 306, 308, 310, 312 or 314, a variant polynucleotide of apolynucleotide encoding an amino acid sequence as shown in SEQ ID NO:322 encodes an amino acid sequence as shown in any one of SEQ ID NOs.:324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350,352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378,380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406,408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434,436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462,464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490,492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518,520 or 522, a variant polynucleotide of a polynucleotide encoding anamino acid sequence as shown in SEQ ID NO: 553 encodes an amino acidsequence as shown in any one of SEQ ID NOs.: 555, 557, 559, 561, 563,565, 567, 569, 571, 573, 575, 577, 579, 581 or 583, a variantpolynucleotide of a polynucleotide encoding an amino acid sequence asshown in SEQ ID NO: 592 encodes an amino acid sequence as shown in anyone of SEQ ID NOs.: 594, 596, 598, 600, 602, 604, 606, 608, 610, 612,614 or 616, a variant polynucleotide of a polynucleotide encoding anamino acid sequence as shown in SEQ ID NO: 626 encodes an amino acidsequence as shown in any one of SEQ ID NOs.: 628, 630, 632, 634 or 636,a variant polynucleotide of a polynucleotide encoding an amino acidsequence as shown in SEQ ID NO: 658 encodes an amino acid sequence asshown in any one of SEQ ID NOs.: 660, 662, 664, 666, 668, 670, 672, 674,676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702,704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730,732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758,760, 762, 764 or 766, and a variant polynucleotide of a polynucleotideencoding an amino acid sequence as shown in SEQ ID NO: 779 encodes anamino acid sequence as shown in any one of SEQ ID NOs.: 781, 783, 785,787, 789, 791, 793, 795, 797, 799, 801, 803, 805 or 807.

Most preferably, variant polynucleotides of the polynucleotide shown inSEQ ID NO: 1 are shown in any one of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47,variant polynucleotides of the polynucleotide shown in SEQ ID NO: 63 areshown in any one of SEQ ID NOs: 65, 67, 69, 71, 73, 75 or 77, variantpolynucleotides of the polynucleotide shown in SEQ ID NO: 87 are shownin any one of SEQ ID NOs: 89, 91, 93, 95, 97, 99, 101, 103, 105, 107,109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135 or137, variant polynucleotides of the polynucleotide shown in SEQ ID NO:158 are shown in any one of SEQ ID NOs: 160, 162, 164, 166, 168, 170,172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198,200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226 or228, variant polynucleotides of the polynucleotide shown in SEQ ID NO:238 are shown in any one of SEQ ID NOs: 240, 242, 244, 246, 248, 250,252, 254, 256, 258, 260, 262, 264 or 266, variant polynucleotides of thepolynucleotide shown in SEQ ID NO: 273 are shown in any one of SEQ IDNOs: 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299,301, 303, 305, 307, 309, 311 or 313, variant polynucleotides of thepolynucleotide shown in SEQ ID NO: 321 are shown in any one of SEQ IDNOs: 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347,349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375,377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403,405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431,433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459,461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487,489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515,517, 519 or 521, variant polynucleotides of the polynucleotide shown inSEQ ID NO: 552 are shown in any one of SEQ ID NOs: 554, 556, 558, 560,562, 564, 566, 568, 570, 572, 574, 576, 578, 580 or 582, variantpolynucleotides of the polynucleotide shown in SEQ ID NO: 591 are shownin any one of SEQ ID NOs: 593, 595, 597, 599, 601, 603, 605, 607, 609,611, 613 or 615, variant polynucleotides of the polynucleotide shown inSEQ ID NO: 625 are shown in any one of SEQ ID NOs: 627, 629, 631, 633 or635, variant polynucleotides of the polynucleotide shown in SEQ ID NO:657 are shown in any one of SEQ ID NOs: 659, 661, 663, 665, 667, 669,671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697,699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725,727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753,755, 757, 759, 761, 763 or 765, and variant polynucleotides of thepolynucleotide shown in SEQ ID NO: 778 are shown in any one of SEQ IDNOs: 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804 or806.

The variant polynucleotides or fragments referred to above, preferably,encode polypeptides retaining at least 10%, at least 20%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80% orat least 90% of the biological activity exhibited by the polypeptideshown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 64, 66, 68, 70, 72, 74, 76, 78,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 159, 161, 163,165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191,193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219,221, 223, 225, 227, 229, 241, 243, 245, 247, 249, 251, 253, 255, 257,259, 261, 263, 265, 267, 274, 276, 278, 280, 282, 284, 286, 288, 290,292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 322, 324,326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352,354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380,382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408,410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436,438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464,466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492,494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520,522, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577,579, 581, 583, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612,614, 616, 626, 628, 630, 632, 634, 636, 658, 660, 662, 664, 666, 668,670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696,698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724,726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752,754, 756, 758, 760, 762, 764, 766, 779, 781, 783, 785, 787, 789, 791,793, 795, 797, 799, 801, 803, 805 or 807. The activity may be tested asdescribed in the accompanying Examples.

The polynucleotides of the present invention either essentially consistof the aforementioned nucleic acid sequences or comprise theaforementioned nucleic acid sequences. Thus, they may contain furthernucleic acid sequences as well. Preferably, the polynucleotide of thepresent invention may comprise in addition to an open reading framefurther untranslated sequence at the 3′ and at the 5′ terminus of thecoding gene region: at least 500, preferably 200, more preferably 100nucleotides of the sequence upstream of the 5′ terminus of the codingregion and at least 100, preferably 50, more preferably 20 nucleotidesof the sequence downstream of the 3′ terminus of the coding gene region.Furthermore, the polynucleotides of the present invention may encodefusion proteins wherein one partner of the fusion protein is apolypeptide being encoded by a nucleic acid sequence recited above. Suchfusion proteins may comprise as additional part other enzymes of thefatty acid or lipid biosynthesis pathways, polypeptides for monitoringexpression (e.g., green, yellow, blue or red fluorescent proteins,alkaline phosphatase and the like) or so called “tags” which may serveas a detectable marker or as an auxiliary measure for purificationpurposes. Tags for the different purposes are well known in the art andcomprise FLAG-tags, 6-histidine-tags, MYC-tags and the like.

Variant polynucleotides as referred to in accordance with the presentinvention may be obtained by various natural as well as artificialsources. For example, polynucleotides may be obtained by in vitro and invivo mutagenesis approaches using the above mentioned specificpolynucleotides as a basis. Moreover, polynucleotides being homologs ororthologs may be obtained from various animal, plant, bacteria or fungusspecies. Paralogs may be identified from Arabidopsis thaliana, Brassicanapus or Glycine max.

The polynucleotide of the present invention shall be provided,preferably, either as an isolated polynucleotide (i.e. isolated from itsnatural context such as a gene locus) or in genetically modified orexogenously (i.e. artificially) manipulated form. An isolatedpolynucleotide can, for example, comprise less than approximately 5 kb,4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences whichnaturally flank the nucleic acid molecule in the genomic DNA of the cellfrom which the nucleic acid is derived. The polynucleotide, preferably,is double or single stranded DNA including cDNA or RNA. The termencompasses single- as well as double-stranded polynucleotides.Moreover, comprised are also chemically modified polynucleotidesincluding naturally occurring modified polynucleotides such asglycosylated or methylated polynucleotides or artificial modified onessuch as biotinylated polynucleotides. Further variant polynucleotidesencompass peptide nucleic acids (PNAs). Such a PNA, preferably,comprises a peptide moiety chemically linked to a polynucleotide havinga nucleic acid sequence of a polynucleotide of the present invention ora fragment thereof.

The polynucleotide encoding a polypeptide having a biological activityas specified encompassed by the present invention is also, preferably, apolynucleotide having a nucleic acid sequence which has been adopted tothe specific codon-usage of the organism, e.g., the plant species, inwhich the polynucleotide shall be expressed (i.e. the target organism).This is, in general, achieved by changing the codons of a nucleic acidsequence obtained from a first organism (i.e. the donor organism)encoding a given amino acid sequence into the codons normally used bythe target organism whereby the amino acid sequence is retained. It isin principle acknowledged that the genetic code is redundant (i.e.degenerated). Specifically, 61 codons are used to encode only 20 aminoacids. Thus, a majority of the 20 amino acids will be encoded by morethan one codon. The codons for the amino acids are well known in the artand are universal to all organisms. However, among the different codonswhich may be used to encode a given amino acid, each organism maypreferably use certain codons. The presence of rarely used codons in anucleic acid sequence will result a depletion of the respective tRNApools and, thereby, lower the translation efficiency. Thus, it may beadvantageous to provide a polynucleotide comprising a nucleic acidsequence encoding a polypeptide as referred to above wherein saidnucleic acid sequence is optimized for expression in the target organismwith respect to the codon usage. In order to optimize the codon usagefor a target organism, a plurality of known genes from the said organismmay be investigated for the most commonly used codons encoding the aminoacids. In a subsequent step, the codons of a nuclei acid sequence fromthe donor organism will be optimized by replacing the codons in thedonor sequence by the codons most commonly used by the target organismfor encoding the same amino acids. It is to be understood that if thesame codon is used preferably by both organisms, no replacement will benecessary. For various target organisms, tables with the preferred codonusages are already known in the art; see e.g.,http://www.kazusa.or.jp/Kodon/E.html. Moreover, computer programs existfor the optimization, e.g., the Leto software, version 1.0 (EntelechonGmbH, Germany) or the GeneOptimizer (Geneart AG, Germany). For theoptimization of a nucleic acid sequence, several criteria may be takeninto account. For example, for a given amino acid, always the mostcommonly used codon may be selected for each codon to be exchanged.Alternatively, the codons used by the target organism may replace thosein a donor sequence according to their naturally frequency. Accordingly,at some positions even less commonly used codons of the target organismwill appear in the optimized nucleic acid sequence. The distribution ofthe different replacement codons of the target organism to the donornucleic acid sequence may be randomly. Preferred target organisms inaccordance with the present invention are soybean or canola (Brassica)species. Preferably, the polynucleotide of the present invention has anoptimized nucleic acid for codon usage in the envisaged target organismwherein at least 20%, at least 40%, at least 60%, at least 80% or all ofthe relevant codons are adopted.

Advantageously, it has been found in the studies underlying the presentinvention that the polypeptides being encoded by the polynucleotides ofthe present invention are capable of increasing the content of seedstorage compounds and, in particular, of lipids or fatty acids, inplants. Thus, the polynucleotides of the present invention are, inprinciple, useful for the synthesis of seed storage compounds such asfatty acids or lipids. Moreover, they may be used to generate transgenicplants or seeds thereof having a modified, preferably increased, amountof seed storage compounds. Such transgenic plants or seeds may be usedfor the manufacture of seed oil or other lipid and/or fatty acidcontaining compositions.

Further, the present invention relates to a vector comprising thepolynucleotide of the present invention. Preferably, the vector is anexpression vector.

The term “vector”, preferably, encompasses phage, plasmid, viral orretroviral vectors as well as artificial chromosomes, such as bacterialor yeast artificial chromosomes. Moreover, the term also relates totargeting constructs which allow for random or site-directed integrationof the targeting construct into genomic DNA. Such target constructs,preferably, comprise DNA of sufficient length for either homolgousrecombination or heterologous insertion as described in detail below.The vector encompassing the polynucleotides of the present invention,preferably, further comprises selectable markers for propagation and/orselection in a host. The vector may be incorporated into a host cell byvarious techniques well known in the art. If introduced into a hostcell, the vector may reside in the cytoplasm or may be incorporated intothe genome. In the latter case, it is to be understood that the vectormay further comprise nucleic acid sequences which allow for homologousrecombination or heterologous insertion, see below. Vectors can beintroduced into prokaryotic or eukaryotic cells via conventionaltransformation or transfection techniques. An “expression vector”according to the present invention is characterized in that it comprisesan expression control sequence such as promoter and/or enhancer sequenceoperatively linked to the polynucleotide of the present invention.Preferred vectors, expression vectors and transformation or transfectiontechniques are specified elsewhere in this specification in detail.

Furthermore, the present invention encompasses a host cell comprisingthe polynucleotide or vector of the present invention.

Host cells are primary cells or cell lines derived from multicellularorganisms such as plants or animals. Furthermore, host cells encompassprokaryotic or eukaryotic single cell organisms (also referred to asmicroorganisms), e.g. bacteria or fungi including yeast or bacteria.Primary cells or cell lines to be used as host cells in accordance withthe present invention may be derived from the multicellular organisms,preferably from plants. Specifically preferred host cells,microorganisms or multicellular organism from which host cells may beobtained are disclosed below.

The polynucleotides or vectors of the present invention may beincorporated into a host cell or a cell of a transgenic non-humanorganism by heterologous insertion or homologous recombination.“Heterologous” as used in the context of the present invention refers toa polynucleotide which is inserted (e.g., by ligation) or is manipulatedto become inserted to a nucleic acid sequence context which does notnaturally encompass the said polynucleotide, e.g., an artificial nucleicacid sequence in a genome of an organism. Thus, a heterologouspolynucleotide is not endogenous to the cell into which it isintroduced, but has been obtained from another cell. Generally, althoughnot necessarily, such heterologous polynucleotides encode proteins thatare normally not produced by the cell expressing the said heterologouspolynucleotide. An expression control sequence as used in a targetingconstruct or expression vector is considered to be “heterologous” inrelation to another sequence (e.g., encoding a marker sequence or anagronomically relevant trait) if said two sequences are either notcombined or operatively linked in a different way in their naturalenvironment. Preferably, said sequences are not operatively linked intheir natural environment (i.e. originate from different genes). Mostpreferably, said regulatory sequence is covalently joined (i.e. ligated)and adjacent to a nucleic acid to which it is not adjacent in itsnatural environment. “Homologous” as used in accordance with the presentinvention relates to the insertion of a polynucleotide in the sequencecontext in which the said polynucleotide naturally occurs. Usually, aheterologous polynucleotide is also incorporated into a cell byhomologous recombination. To this end, the heterologous polynucleotideis flanked by nucleic acid sequences being homologous to a targetsequence in the genome of a host cell or a non-human organism.Homologous recombination now occurs between the homologous sequences.However, as a result of the homologous recombination of the flankingsequences, the heterologous polynucleotide will be inserted, too. How toprepare suitable target constructs for homologous recombination and howto carry out the said homologous recombination is well known in the art.

Also provided in accordance with the present invention is a method forthe manufacture of a polypeptide being capable of increasing the seedstorage compound content when expressed in transgenic plants comprising:

(a) expressing the polynucleotide of the present invention in a hostcell; and

(b) obtaining the polypeptide encoded by said polynucleotide from thehost cell.

The polypeptide may be obtained, for example, by all conventionalpurification techniques including affinity chromatography, sizeexclusion chromatography, high pressure liquid chromatography (HPLC) andprecipitation techniques including antibody precipitation. It is to beunderstood that the method may—although preferred—not necessarily yieldan essentially pure preparation of the polypeptide. It is to beunderstood that depending on the host cell which is used for theaforementioned method, the polypeptides produced thereby may becomeposttranslationally modified or processed otherwise.

The present invention, moreover, pertains to a polypeptide encoded bythe polynucleotide of the present invention or which is obtainable bythe aforementioned method of the present invention.

The term “polypeptide” as used herein encompasses essentially purifiedpolypeptides or polypeptide preparations comprising other proteins inaddition. Further, the term also relates to the fusion proteins orpolypeptide fragments being at least partially encoded by thepolynucleotide of the present invention referred to above. Moreover, itincludes chemically modified polypeptides. Such modifications may beartificial modifications or naturally occurring modifications such asphosphorylation, glycosylation, myristylation and the like. The terms“polypeptide”, “peptide” or “protein” are used interchangeablethroughout this specification. The polypeptide of the present inventionshall exhibit the biological activities referred to above and, morepreferably, it shall be capable of increasing the amount of seed storagecompounds, preferably, fatty acids or lipids, when present in plantseeds as referred to above. Most preferably, if present in plant seeds,the polypeptide shall be capable of significantly increasing the seedstorage of fatty acids as described in the accompanying Examples below.

Encompassed by the present invention is, furthermore, an antibody whichspecifically recognizes the polypeptide of the invention.

Antibodies against the polypeptides of the invention can be prepared bywell known methods using a purified polypeptide according to theinvention or a suitable fragment derived therefrom as an antigen. Afragment which is suitable as an antigen may be identified byantigenicity determining algorithms well known in the art. Suchfragments may be obtained either from the polypeptide of the inventionby proteolytic digestion or may be a synthetic peptide. Preferably, theantibody of the present invention is a monoclonal antibody, a polyclonalantibody, a single chain antibody, a human or humanized antibody orprimatized, chimerized or fragment thereof. Also comprised as antibodiesby the present invention are a bispecific antibody, a syntheticantibody, an antibody fragment, such as Fab, Fv or scFv fragments etc.,or a chemically modified derivative of any of these. The antibody of thepresent invention shall specifically bind (i.e. does significantly notcross react with other polypeptides or peptides) to the polypeptide ofthe invention. Specific binding can be tested by various well knowntechniques. Antibodies or fragments thereof can be obtained by usingmethods which are described, e.g., in Harlow and Lane “Antibodies, ALaboratory Manual”, CSH Press, Cold Spring Harbor, 1988. Monoclonalantibodies can be prepared by the techniques originally described inKöhler and Milstein, Nature 256 (1975), 495, and Galfré, Meth. Enzymol.73 (1981), 3, which comprise the fusion of mouse myeloma cells to spleencells derived from immunized mammals. The antibodies can be used, forexample, for the immunoprecipitation, immunolocalization or purification(e.g., by affinity chromatography) of the polypeptides of the inventionas well as for the monitoring of the presence of said variantpolypeptides, for example, in recombinant organisms, and for theidentification of compounds interacting with the proteins according tothe invention.

The present invention also relates to a transgenic non-human organismcomprising the polynucleotide, the vector or the host cell of thepresent invention. Preferably, said non-human transgenic organism is aplant.

The term “non-human transgenic organism”, preferably, relates to aplant, an animal or a multicellular microorganism. The polynucleotide orvector may be present in the cytoplasm of the organism or may beincorporated into the genome either heterologous or by homologousrecombination. Host cells, in particular those obtained from plants oranimals, may be introduced into a developing embryo in order to obtainmosaic or chimeric organisms, i.e. non-human transgenic organismscomprising the host cells of the present invention. Preferably, thenon-human transgenic organism expresses the polynucleotide of thepresent invention in order to produce the polypeptide in an amountresulting in a detectable biological activity as specified above.Suitable transgenic organisms are, preferably, all those organisms whichare capable of synthesizing fatty acids or lipids. Preferred organismsand methods for transgenesis are disclosed in detail below. A transgenicorganism or tissue may comprise one or more transgenic cells.Preferably, the organism or tissue is substantially consisting oftransgenic cells (i.e., more than 80%, preferably 90%, more preferably95%, most preferably 99% of the cells in said organism or tissue aretransgenic). The term “transgene” as used herein refers to any nucleicacid sequence, which is introduced into the genome of a cell or whichhas been manipulated by experimental manipulations including techniquessuch as chimeraplasty or genoplasty. Preferably, said sequence isresulting in a genome which is significantly different from the overallgenome of an organism (e.g., said sequence, if endogenous to saidorganism, is introduced into a location different from its naturallocation, or its copy number is increased or decreased). A transgene maycomprise an endogenous polynucleotide (i.e. a polynucleotide having anucleic acid sequence obtained from the same organism or host cell) ormay be obtained from a different organism or hast cell, wherein saiddifferent organism is, preferably an organism of another species and thesaid different host cell is, preferably, a different microorganism, ahost cell of a different origin or derived from a an organism of adifferent species.

Particularly preferred as a plant to be used in accordance with thepresent invention are oil producing plant species. Most preferably, thesaid plant is selected from the group consisting of canola, linseed,soybean, sunflower, maize, oat, rye, barley, wheat, rice, pepper,tagetes, cotton, oil palm, coconut palm, flax, castor and peanut,

The present invention relates to a method for the manufacture of a lipidand/or a fatty acid comprising the steps of:

-   (a) cultivating (i) the host cell or the transgenic non-human    organism of the present invention or (ii) a host cell or a non-human    transgenic organism comprising a polynucleotide comprising a nucleic    acid sequence as shown in any one of SEQ ID NO: 139, 141, 143, 145,    147, 523, 525, 527, 529, 531, 533, 535, 537, 539, 584, 617, 619,    637, 639, 641, 767, 769, 808, 810, 812 or 814 or encoding an amino    acid sequence as shown in any one of SEQ ID NOs: 140, 142, 144, 146,    148, 524, 526, 528, 530, 532, 534, 536, 538, 540, 585, 618, 620,    638, 640, 642, 768, 770, 809, 811, 813 or 815 under conditions    allowing synthesis of the said lipid or fatty acid; an-   (b) obtaining the said lipid and/or fatty acid from the host cell or    the transgenic non-human organism.

The term “lipid” and “fatty acid” as used herein refer, preferably, tothose recited in Table 13 (for lipids) and Table 14 (for fatty acids),below. However, the terms, in principle, also encompass other lipids orfatty acids which can be obtained by the lipid metabolism in a host cellor an organism referred to in accordance with the present invention.

A host cell or a non-human transgenic organism expressing apolynucleotide comprising a nucleic acid sequence as shown in SEQ ID NO:139, 141, 143, 145, 147, 523, 525, 527, 529, 531, 533, 535, 537, 539,584, 617, 619, 637, 639, 641, 767, 769, 808, 810, 812 or 814 can beobtained by any of the insertion or recombination techniques referred toelsewhere in this specification. It is, preferably, envisaged that thepolynucleotide is a heterologous polynucleotide with respect to the hostcell or the non-human organism. The polynucleotides comprising a nucleicacid sequence as shown in SEQ ID NO: 139, 141, 143, 145, 147, 523, 525,527, 529, 531, 533, 535, 537, 539, 584, 617, 619, 637, 639, 641, 767,769, 808, 810, 812 or 814 encode a polypeptide having an amino acidsequence as shown in SEQ ID NO: 140, 142, 144, 146, 148, 524, 526, 528,530, 532, 534, 536, 538, 540, 585, 618, 620, 638, 640, 642, 768, 770,809, 811, 813 or 815. These sequences represent distantly relatedhomologs. However, it has been found that these sequences are alsocapable to modify and, preferably, increase the amount of seed storagecompounds in plants. Accordingly, these polynucleotides as well asvariants may be also used in the methods of the present inventionalthough less efficiently. The definition of the term “variant” made inconnection with the polynucleotides of the present invention appliesmutatis mutandis for the variants of the aforementioned polynucleotides(i.e. SEQ ID NOs: 139, 141, 143, 145, 147, 523, 525, 527, 529, 531, 533,535, 537, 539, 584, 617, 619, 637, 639, 641, 767, 769, 808, 810, 812 or814).

In a preferred embodiment of the aforementioned method of the presentinvention, the said lipid and/or fatty acids constitute seed oil.

Moreover, the present invention pertains to a method for the manufactureof a plant having a modified amount of a seed storage compound,preferably a lipid or a fatty acid, comprising the steps of:

-   (a) introducing (i) the polynucleotide or the vector of the present    invention or (ii) a polynucleotide comprising a nucleic acid    sequence as shown in any one of SEQ ID NO: 139, 141, 143, 145, 147,    523, 525, 527, 529, 531, 533, 535, 537, 539, 584, 617, 619, 637,    639, 641, 767, 769, 808, 810, 812 or 814 or encoding an amino acid    sequence as shown in any one of SEQ ID NOs: 140, 142, 144, 146, 148,    524, 526, 528, 530, 532, 534, 536, 538, 540, 585, 618, 620, 638,    640, 642, 768, 770, 809, 811, 813 or 815 into a plant cell; and-   (b) generating a transgenic plant from the said plant cell, wherein    the polypeptide encoded by the polynucleotide modifies the amount of    the said seed storage compound in the transgenic plant.

The term “seed storage compound” as used herein, preferably, refers tocompounds being a sugar, a protein, or, more preferably, a lipid or afatty acid. Preferably, the amount of said seed storage compound issignificantly increased compared to a control, preferably an emptyvector control as specified above. The increase is, more preferably, anincrease in the amount by weight of at least 1, 2.5, 5, 7.5, 10, 12.5,15, 17.5, 20, 22.5 or 25% as compared to a control.

It is to be understood that the polynucleotides or the vector referredto in accordance with the above method of the present invention may beintroduced into the plant cell by any of the aforementioned insertion orrecombination techniques.

The aforementioned method of the present invention may be also used tomanufacture a plant having an altered total oil content in its seeds ora plant having an altered total seed oil content and altered levels ofseed storage compounds in its seeds. Such plants are suitable sourcesfor seed oil and may be used for the large scale manufacture thereof.

Further preferred embodiments of the compounds, methods and usesaccording to the present invention are described in the following.Moreover, the terms used above will be explained in more detail. Thepolynucleotides and polypeptides of the present invention are alsoreferred to as Lipid Metabolism proteins (LMP) herein below.

The present invention also provides an isolated nucleic acid fromArabidopsis thaliana encoding a Lipid Metabolism Protein (LMP), or aportion thereof. These sequences may be used to modify or increaselipids and fatty acids, cofactors, and enzymes in microorganisms andplants.

Arabidopsis plants are known to produce considerable amounts of fattyacids, like linoleic and linolenic acid (see, e.g., Table 14), and fortheir close similarity in many aspects (gene homology etc.) to the oilcrop plant Brassica. Therefore, nucleic acid molecules originating froma plant like Arabidopsis thaliana, Brassica napus, Glycine max, Zeamays, Oryza sativa, Hordeum vulgare, Linum usitatissimum, Triticumaestivum, Helianthus anuus, Beta vulgaris, or Physcomitrella patens, orrelated organisms, are especially suited to modify the lipid and fattyacid metabolism in a host, especially in microorganisms and plants.Furthermore, nucleic acids from the plant Arabidopsis thaliana, Brassicanapus, Glycine max, Zea mays, Oryza sativa, Hordeum vulgare, Linumusitatissimum, Triticum aestivum, Helianthus anuus, Beta vulgaris orPhyscomitrella patens, or related organisms, can be used to identifythose DNA sequences and enzymes in other species, which are useful tomodify the biosynthesis of precursor molecules of fatty acids in therespective organisms.

The present invention further provides an isolated nucleic acidcomprising a fragment of at least 15 nucleotides of a nucleic acid froma plant (Arabidopsis thaliana) encoding an LMP or a portion thereof.

The present invention, thus, also encompasses an oligonucleotide whichspecifically binds to the polynucleotides of the present invention.Binding as meant in this context refers to hybridization by Watson-Crickbase pairing discussed elsewhere in the specification in detail. Anoligonucleotide as used herein has a length of at most 100, at most 50,at most 40, at most 30 or at most 20 nucleotides in length which arecomplementary to the nucleic acid sequence of the polynucleotides of thepresent invention. The sequence of the oligonucleotide is, preferably,selected so that a perfect match by Watson-Crick base pairing will beobtained. The oligonucleotides of the present invention may be suitableas primers for PCR-based amplification techniques. Moreover, theoligonucleotides may be used for RNA interference (RNAi) approaches inorder to modulate and, preferably down-regulate, the activity of thepolypeptides encoded by the polynucleotides of the present invention.Thereby, an organism may be depleted of fatty acids and/or lipids and,specifically, a plant seed may be depleted of at least some of its seedstorage compounds. As used herein, the term “RNA interference (RNAi)”refers to selective intracellular degradation of RNA used to silenceexpression of a selected target gene, i.e. the polynucleotide of thepresent invention. RNAi is a process of sequence-specific,post-transcriptional gene silencing in organisms initiated bydouble-stranded RNA (dsRNA) that is homologous in sequence to the geneto be silenced. The RNAi technique involves small interfering RNAs(siRNAs) that are complementary to target RNAs (encoding a gene ofinterest) and specifically destroy the known mRNA, thereby diminishingor abolishing gene expression. RNAi is generally used to silenceexpression of a gene of interest by targeting mRNA, however, any type ofRNA is encompassed by the RNAi methods of the invention. Briefly, theprocess of RNAi in the cell is initiated by long double stranded RNAs(dsRNAs) being cleaved by a ribonuclease, thus producing siRNA duplexes.The siRNA binds to another intracellular enzyme complex which is therebyactivated to target whatever mRNA molecules are homologous (orcomplementary) to the siRNA sequence. The function of the complex is totarget the homologous mRNA molecule through base pairing interactionsbetween one of the siRNA strands and the target mRNA. The mRNA is thencleaved approximately 12 nucleotides from the 3′ terminus of the siRNAand degraded. In this manner, specific mRNAs can be targeted anddegraded, thereby resulting in a loss of protein expression from thetargeted mRNA. A complementary nucleotide sequence as used herein refersto the region on the RNA strand that is complementary to an RNAtranscript of a portion of the target gene. The term “dsRNA” refers toRNA having a duplex structure comprising two complementary andanti-parallel nucleic acid strands. Not all nucleotides of a dsRNAnecessarily exhibit complete Watson-Crick base pairs; the two RNAstrands may be substantially complementary. The RNA strands forming thedsRNA may have the same or a different number of nucleotides, with themaximum number of base pairs being the number of nucleotides in theshortest strand of the dsRNA. Preferably, the dsRNA is no more than 49,more preferably less than 25, and most preferably between 19 and 23,nucleotides in length. dsRNAs of this length are particularly efficientin inhibiting the expression of the target gene using RNAi techniques.dsRNAs are subsequently degraded by a ribonuclease enzyme into shortinterfering RNAs (siRNAs). RNAi is mediated by small interfering RNAs(siRNAs). The term “small interfering RNA” or “siRNA” refers to anucleic acid molecule which is a double stranded RNA agent that iscomplementary to i.e., able to base-pair with, a portion of a target RNA(generally mRNA), i.e. the polynucleotide of the present invention beingRNA. siRNA acts to specifically guide enzymes in the host cell to cleavethe target RNA. By virtue of the specificity of the siRNA sequence andits homology to the RNA target, siRNA is able to cause cleavage of thetarget RNA strand, thereby inactivating the target RNA molecule.Preferably, the siRNA which is sufficient to mediate RNAi comprises anucleic acid sequence comprising an inverted repeat fragment of thetarget gene and the coding region of the gene of interest (or portionthereof). Also preferably, a nucleic acid sequence encoding a siRNAcomprising a sequence sufficiently complementary to a target gene isoperatively linked to a expression control sequence. Thus, the mediationof RNAi to inhibit expression of the target gene can be modulated bysaid expression control sequence. Preferred expression control sequencesare those which can be regulated by a exogenous stimulus, such as thetet operator whose activity can be regulated by tetracycline or heatinducible promoters. Alternatively, an expression control sequence maybe used which allows tissue-specific expression of the siRNA. Thecomplementary regions of the siRNA allow sufficient hybridization of thesiRNA to the target RNA and thus mediate RNAi. In mammalian cells,siRNAs are approximately 21-25 nucleotides in length (see Tuschl et al.1999 and Elbashir et al. 2001). The siRNA sequence needs to be ofsufficient length to bring the siRNA and target RNA together throughcomplementary base-pairing interactions. The siRNA used with the Tetexpression system of the invention may be of varying lengths. The lengthof the siRNA is preferably greater than or equal to ten nucleotides andof sufficient length to stably interact with the target RNA;specifically 15-30 nucleotides; more specifically any integer between 15and 30 nucleotides, most preferably 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, and 30. By “sufficient length” is meant anoligonucleotide of greater than or equal to 15 nucleotides that is of alength great enough to provide the intended function under the expectedcondition. By “stably interact” is meant interaction of the smallinterfering RNA with target nucleic acid (e.g., by forming hydrogenbonds with complementary nucleotides in the target under physiologicalconditions). Generally, such complementarity is 100% between the siRNAand the RNA target, but can be less if desired, preferably 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99%. For example, 19 bases out of 21bases may be base-paired. In some instances, where selection betweenvarious allelic variants is desired, 100% complementary to the targetgene is required in order to effectively discern the target sequencefrom the other allelic sequence. When selecting between allelic targets,choice of length is also an important factor because it is the otherfactor involved in the percent complementary and the ability todifferentiate between allelic differences. Methods relating to the useof RNAi to silence genes in organisms, including C. elegans, Drosophila,plants, and mammals, are known in the art (see, for example, Fire etal., Nature (1998) 391:806-811; Fire, Trends Genet. 15, 358-363 (1999);Sharp, RNA interference 2001. Genes Dev. 15, 485-490 (2001); Hammond etal. Nature Rev. Genet. 2, 1110-1119 (2001); Tuschl, Chem. Biochem. 2,239-245 (2001); Hamilton et al., Science 286, 950-952 (1999); Hammond etal., Nature 404, 293-296 (2000); Zamore et al., Cell 101, 25-33 (2000);Bernstein et al., Nature 409, 363-366 (2001); Elbashir et al., GenesDev. 15, 188-200 (2001); WO 0129058; WO 09932619; and Elbashir et al.,2001 Nature 411: 494-498).

Also provided by the present invention are polypeptides encoded by thenucleic acids, and heterologous polypeptides comprising polypeptidesencoded by the nucleic acids, and antibodies to those polypeptides.

Additionally, the present invention relates to, and provides the use of,LMP nucleic acids in the production of transgenic plants having amodified level or composition of a seed storage compound. In regard toan altered composition, the present invention can be used, for example,to increase the percentage of oleic acid relative to other plant oils. Amethod of producing a transgenic plant with a modified level orcomposition of a seed storage compound includes the steps oftransforming a plant cell with an expression vector comprising an LMPnucleic acid and generating a plant with a modified level or compositionof the seed storage compound from the plant cell. In a preferredembodiment, the plant is an oil-producing species selected from thegroup consisting of, for example, canola, linseed, soybean, sunflower,maize, oat, rye, barley, wheat, rice, pepper, tagetes, cotton, oil palm,coconut palm, flax, castor, and peanut.

According to the present invention, the compositions and methodsdescribed herein can be used to alter the composition of an LMP in atransgenic plant and to increase or decrease the level of an LMP in atransgenic plant comprising increasing or decreasing the expression ofan LMP nucleic acid in the plant. Increased or decreased expression ofthe LMP nucleic acid can be achieved through transgenic overexpression,co-suppression approaches, antisense approaches, and in vivo mutagenesisof the LMP nucleic acid. The present invention can also be used toincrease or decrease the level of a lipid in a seed oil, to increase ordecrease the level of a fatty acid in a seed oil, or to increase ordecrease the level of a starch in a seed or plant.

More specifically, the present invention includes, and provides a methodfor, altering (increasing or decreasing or changing the specific profileof) the total oil content in a seeds comprising: Transforming a plantwith a nucleic acid construct that comprises, as operably-linkedcomponents, a promoter and nucleic acid sequences capable of modulatingthe level of LMP mRNA or LMP protein, and growing the plant.Furthermore, the present invention includes, and provides a method for,altering (increasing or decreasing) the level of oleic acid in a seedcomprising: Transforming a plant with a nucleic acid construct thatcomprises as operably linked components, a promoter, a structuralnucleic acid sequence capable of altering (increasing or decreasing) thelevel of oleic acid, and growing the plant.

Also included herein is a seed produced by a transgenic planttransformed by an LMP DNA sequence, wherein the seed contains the LMPDNA sequence, and wherein the plant is true breeding for a modifiedlevel of a seed-storage compound. The present invention additionallyincludes a seed oil produced by the aforementioned seed.

Further provided by the present invention are vectors comprising thenucleic acids, host cells containing the vectors, and descendent plantmaterials produced by transforming a plant cell with the nucleic acidsand/or vectors.

According to the present invention, the compounds, compositions, andmethods described herein can be used to increase or decrease therelative percentages of a lipid in a seed oil, increase or decrease thelevel of a lipid in a seed oil, or to increase or decrease the level ofa fatty acid in a seed oil, or to increase or decrease the level of astarch or other carbohydrate in a seed or plant, or to increase ordecrease the level of proteins in a seed or plant. The manipulationsdescribed herein can also be used to improve seed germination and growthof the young seedlings and plants and to enhance plant yield of seedstorage compounds.

It is further provided a method of producing a higher or lower thannormal or typical level of storage compound in a transgenic plantexpressing an LMP nucleic acid from Arabidopsis thaliana, Brassicanapus, Glycine max and Oryza sativa in the transgenic plant, wherein thetransgenic plant is Arabidopsis thaliana, Brassica napus, Glycine max,Oryza sativa, Zea mays, Triticum aestivum, Hordeum vulgare, Linumusitatissimum, Helianthus anuus, or Beta vulgaris or a species differentfrom Arabidopsis thaliana, Brassica napus, Glycine max and Oryza sativa.Also included herein are compositions and methods of the modification ofthe efficiency of production of a seed storage compound. As used herein,where the phrase Arabidopsis thaliana, Brassica napus, Glycine max,Oryza sativa, Zea mays, Triticum aestivum, Hordeum vulgare, Linumusitatissimum, Helianthus anuus, or Beta vulgaris is used, this alsomeans Arabidopsis thaliana and/or Brassica napus and/or Glycine maxand/or Oryza sativa and/or Zea mays and/or Triticum aestivum and/orHordeum vulgare and/or Linum usitatissimum and/or Helianthus anuusand/or Beta vulgaris.

Accordingly, it is an object of the present invention to provide novelisolated LMP nucleic acids and isolated LMP amino acid sequences fromArabidopsis thaliana, Brassica napus, Glycine max and Oryza sativa, aswell as active fragments, analogs, and orthologs thereof. Those activefragments, analogs, and orthologs can also be from different plantspecies, as one skilled in the art will appreciate that other plantspecies will also contain those or related nucleic acids.

It is another object of the present invention to provide transgenicplants having modified levels of seed storage compounds, and, inparticular, modified levels of a lipid, a fatty acid, or a sugar.

The polynucleotides and polypeptides of the present invention, includingagonists and/or fragments thereof, have also uses that includemodulating plant growth, and potentially plant yield, preferablyincreasing plant growth under adverse conditions (drought, cold, light,UV). In addition, antagonists of the present invention may have usesthat include modulating plant growth and/or yield through preferablyincreasing plant growth and yield. In yet another embodiment,over-expression polypeptides of the present invention, using aconstitutive promoter, may be useful for increasing plant yield understress conditions (drought, light, cold, UV) by modulatinglight-utilization efficiency. Moreover, polynucleotides and polypeptidesof the present invention will improve seed germination and seed dormancyand, hence, will improve plant growth and/or yield of seed storagecompounds.

The isolated nucleic acid molecules of the present invention may furthercomprise an operably linked promoter or partial promoter region. Thepromoter can be a constitutive promoter, an inducible promoter, or atissue-specific promoter. The constitutive promoter can be, for example,the superpromoter (Ni et al., Plant J. 7:661-676, 1995; U.S. Pat. No.5,955,646). The tissue-specific promoter can be active in vegetativetissue or reproductive tissue. The tissue-specific promoter active inreproductive tissue can be a seed-specific promoter. The tissue-specificpromoter active in vegetative tissue can be a root-specific,shoot-specific, meristem-specific, or leaf-specific promoter. Theisolated nucleic acid molecule of the present invention can stillfurther comprise a 5′ non-translated sequence, 3′ non-translatedsequence, introns, or the combination thereof.

The present invention also provides a method for increasing the numberand/or size or density of one or more plant organs of a plant expressingan isolated nucleic acid from Arabidopsis thaliana, Brassica napus,Glycine max and Oryza sativa encoding a LMP, or a portion thereof. Morespecifically, seed size, and/or seed number and/or weight, might bemanipulated. Moreover, root length or density can be increased. Longeror denser roots can alleviate not only the effects of water depletionfrom soil but also improve plant anchorage/standability, thus reducinglodging. Also, longer or denser roots have the ability to cover a largervolume of soil and improve nutrient uptake. All of these advantages ofaltered root architecture have the potential to increase crop yield.Additionally, the number and size of leaves might be increased by thenucleic acid sequences provided in this application. This will have theadvantage of improving photosynthetic light-utilization efficiency byincreasing photosynthetic light-capture capacity and photosyntheticefficiency.

It is a further object of the present invention to provide methods forproducing such aforementioned transgenic plants.

It is another object of the present invention to provide seeds and seedoils from such aforementioned transgenic plants.

Before the present compounds, compositions, and methods are disclosedand described, it is to be understood that this invention is not limitedto specific nucleic acids, specific polypeptides, specific cell types,specific host cells, specific conditions, or specific methods, etc., assuch may, of course, vary, and the numerous modifications and variationstherein will be apparent to those skilled in the art. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting. As used in the specification and in the claims, “a” or “an”can mean one or more, depending upon the context in which it is used.Thus, for example, reference to “a cell” can mean that at least one cellcan be utilized.

The present invention is based, in part, on the isolation andcharacterization of nucleic acid molecules encoding LMPs from plantsincluding Arabidopsis thaliana, Brassica napus, Glycine max, Oryzasativa, and other related crop species like maize, wheat, rice, barley,linseed, sugar beat, or sunflower.

In accordance with the purpose(s) of this invention, as embodied andbroadly described herein, this invention, in one aspect, provides anisolated nucleic acid from a plant (Arabidopsis thaliana, Brassicanapus, Glycine max, or Oryza sativa) encoding a LMP or a portionthereof.

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode the LMP polypeptides of the present invention, orbiologically active portions thereof, as well as nucleic acid fragmentssufficient for use as hybridization probes or primers for theidentification or amplification of an LMP-encoding nucleic acid (e.g.,LMP DNA). As used herein, the term “nucleic acid molecule” is intendedto include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules(e.g., mRNA) and analogs of the DNA or RNA generated using nucleotideanalogs. This term also encompasses untranslated sequence located atboth the 3′ and 5′ ends of the coding region of a gene: At least about1000 nucleotides of sequence upstream from the 5′ end of the codingregion and at least about 200 nucleotides of sequence downstream fromthe 3′ end of the coding region of the gene. The nucleic acid moleculecan be single-stranded or double-stranded, but preferably isdouble-stranded DNA. An “isolated” nucleic acid molecule is one that issubstantially separated from other nucleic acid molecules, which arepresent in the natural source of the nucleic acid. Preferably, an“isolated” nucleic acid is substantially free of sequences thatnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For example, in various embodiments,the isolated LMP nucleic acid molecule can contain less than about 5 kb,4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences, whichnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived (e.g., an Arabidopsis thaliana,Brassica napus, Glycine max, Oryza sativa cell). Moreover, an “isolated”nucleic acid molecule, such as a cDNA molecule, can be substantiallyfree of other cellular material, or culture medium, when produced byrecombinant techniques or chemical precursors, or other chemicals whenchemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having a nucleotide sequence as specified elsewhere in thedescription, or a portion thereof, can be isolated using standardmolecular biology techniques and the sequence information providedherein. For example, an Arabidopsis thaliana, Brassica napus, Glycinemax or Oryza sativa LMP cDNA can be isolated from an Arabidopsisthaliana, Brassica napus, Glycine max or Oryza sativa library using allor portion of one of the specific sequences disclosed herein as ahybridization probe and standard hybridization techniques (e.g., asdescribed in Sambrook et al. 1989, Molecular Cloning: A LaboratoryManual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.). Moreover, a nucleic acidmolecule encompassing all or a portion of one of the specific sequencesdisclosed herein can be isolated by the polymerase chain reaction usingoligonucleotide primers designed based upon this sequence (e.g., anucleic acid molecule encompassing all or a portion of one of thespecific sequences disclosed herein can be isolated by the polymerasechain reaction using oligonucleotide primers designed based upon thissame sequence). For example, mRNA can be isolated from plant cells(e.g., by the guanidinium-thiocyanate extraction procedure of Chirgwinet al. 1979, Biochemistry 18:5294-5299) and cDNA can be prepared usingreverse transcriptase (e.g., Moloney MLV reverse transcriptase,available from Gibco/BRL, Bethesda, Md.; or AMV reverse transcriptase,available from Seikagaku America, Inc., St. Petersburg, Fla.). Syntheticoligonucleotide primers for polymerase chain reaction amplification canbe designed based upon one of the specific sequences disclosed herein. Anucleic acid of the invention can be amplified using cDNA or,alternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to an LMP nucleotidesequence can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid of the inventioncomprises one of the nucleotide sequences referred to above. Thespecific sequences disclosed herein correspond to the Arabidopsisthaliana, Brassica napus, Glycine max and Oryza sativa LMP cDNAs of theinvention. These cDNAs comprise sequences encoding LMPs (i.e., the“coding region”, indicated in the table below), as well as 5′untranslated sequences and 3′ untranslated sequences. Alternatively, thenucleic acid molecules can comprise only the coding region of any of thespecific sequences disclosed herein or can contain whole genomicfragments isolated from genomic DNA.

For the purposes of this application, it will be understood that each ofthe specific sequences set forth herein has an identifying entry number(e.g., pk321AT01). Each of these sequences may generally comprise threeparts: a 5′ upstream region, a coding region, and a downstream region. Acoding region of these sequences is indicated as “ORF position” (Table15).

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule, which is a complementof one of the specific nucleotide sequences disclosed herein. A nucleicacid molecule, which is complementary to one of the nucleotide sequencesis one which is sufficiently complementary to the said nucleotidesequence such that it can hybridize by forming a stable duplex.

In still another preferred embodiment, an isolated nucleic acid moleculeof the invention comprises a nucleotide sequence that is at least about50-60%, preferably at least about 60-70%, more preferably at least about70-80%, 80-90%, or 90-95%, and even more preferably at least about 95%,96%, 97%, 98%, 99%, or more homologous to a nucleotide sequence of thepresent invention, or a portion thereof. In an additional preferredembodiment, an isolated nucleic acid molecule of the invention comprisesa nucleotide sequence that hybridizes, e.g., hybridizes under stringentconditions, to one of the nucleotide sequences of the present invention,or a portion thereof. These hybridization conditions include washingwith a solution having a salt concentration of about 0.02 molar at pH 7at about 60° C.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the coding region of one of the sequences of the presentinvention, for example, a fragment that can be used as a probe orprimer, or a fragment encoding a biologically-active portion of an LMP.The nucleotide sequences determined from the cloning of the LMP genesfrom Arabidopsis thaliana, Brassica napus, Glycine max and Oryza sativaallows for the generation of probes and primers designed for use inidentifying and/or cloning LMP homologues in other cell types andorganisms, as well as LMP homologues from other plants or relatedspecies. Therefore, this invention also provides compounds comprisingthe nucleic acids disclosed herein or fragments thereof. These compoundsinclude the nucleic acids attached to a moiety. These moieties include,but are not limited to, detection moieties, hybridization moieties,purification moieties, delivery moieties, reaction moieties, bindingmoieties, and the like. The probe/primer typically comprisessubstantially purified oligonucleotide. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, preferably about 25, morepreferably about 40, 50 or 75 consecutive nucleotides of a sense strandof one of the sequences set forth herein, an anti-sense sequence of oneof the sequences set forth in herein, or naturally-occurring mutantsthereof. Primers based on a nucleotide sequence of the present inventioncan be used in PCR reactions to clone LMP homologues. Probes based onthe LMP nucleotide sequences can be used to detect transcripts orgenomic sequences encoding the same or homologous proteins. In preferredembodiments, the probe further comprises a label group attached thereto,e.g. the label group can be a radioisotope, a fluorescent compound, anenzyme, or an enzyme co-factor. Such probes can be used as a part of agenomic marker test kit for identifying cells that express an LMP, suchas by measuring a level of an LMP-encoding nucleic acid in a sample ofcells, e.g., detecting LMP mRNA levels or determining whether a genomicLMP gene has been mutated or deleted.

In one embodiment, the nucleic acid molecule of the invention encodes aprotein or portion thereof, which includes an amino acid sequence thatis sufficiently homologous to an amino acid encoded by a sequence of thepresent invention, such that the protein or portion thereof maintainsthe same or a similar function as the wild-type protein. As used herein,the language “sufficiently homologous” refers to proteins, or portionsthereof, which have amino acid sequences that include a minimum numberof identical or equivalent (e.g., an amino acid residue that has asimilar side chain as an amino acid residue in one of the ORFs of asequence of the present invention) amino acid residues to an amino acidsequence, such that the protein or portion thereof is able toparticipate in the metabolism of compounds necessary for the productionof seed storage compounds in plants, construction of cellular membranesin microorganisms, or plants, or in the transport of molecules acrossthese membranes. Regulatory proteins, such as DNA binding proteins,transcription factors, kinases, phosphatases, or protein members ofmetabolic pathways, such as the lipid, starch and protein biosyntheticpathways, or membrane transport systems, may play a role in thebiosynthesis of seed storage compounds. Examples of such activities aredescribed herein (see putative annotations in Table 15). Examples ofLMP-encoding nucleic acid sequences of the present invention.

As altered or increased sugar and/or fatty acid production is a generaltrait wished to be inherited into a wide variety of plants like maize,wheat, rye, oat, triticale, rice, barley, soybean, peanut, cotton,canola, manihot, pepper, sunflower, sugar beet and tagetes, solanaceousplants like potato, tobacco, eggplant, and tomato, Vicia species, pea,alfalfa, bushy plants (coffee, cacao, tea), Salix species, trees (oilpalm, coconut), and perennial grasses and forage crops, these cropplants are also preferred target plants for genetic engineering as onefurther embodiment of the present invention.

Portions of proteins encoded by the LMP nucleic acid molecules of theinvention are preferably biologically-active portions of one of theLMPs. As used herein, the term “biologically active portion of an LMP”is intended to include a portion, e.g., a domain/motif, of an LMP thatparticipates in the metabolism of compounds necessary for thebiosynthesis of seed storage lipids, or the construction of cellularmembranes in microorganisms or plants, or in the transport of moleculesacross these membranes, or has an activity as set forth in Table 15. Todetermine whether an LMP, or a biologically active portion thereof, canparticipate in the metabolism of compounds necessary for the productionof seed storage compounds and cellular membranes, an assay of enzymaticactivity may be performed. Such assay methods are well known to thoseskilled in the art, and as described in Example 14 of theExemplification.

Biologically-active portions of an LMP include peptides comprising aminoacid sequences derived from the amino acid sequence of an LMP (e.g., anamino acid sequence encoded by a nucleic acid of the present inventionor the amino acid sequence of a protein homologous to an LMP, whichinclude fewer amino acids than a full length LMP or the full lengthprotein which is homologous to an LMP) and exhibit at least one activityof an LMP. Typically, biologically active portions (peptides, e.g.,peptides which are, for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39,40, 50, 100, or more amino acids in length) comprise a domain or motifwith at least one activity of an LMP. Moreover, other biologicallyactive portions, in which other regions of the protein are deleted, canbe prepared by recombinant techniques and evaluated for one or more ofthe activities described herein. Preferably, the biologically activeportions of an LMP include one or more selected domains/motifs orportions thereof having biological activity.

Additional nucleic acid fragments encoding biologically-active portionsof an LMP can be prepared by isolating a portion of one of thesequences, expressing the encoded portion of the LMP or peptide (e.g.,by recombinant expression in vitro), and assessing the activity of theencoded portion of the LMP or peptide.

The invention further encompasses nucleic acid molecules that differfrom one of the nucleotide sequences of the present invention (andportions thereof) due to degeneracy of the genetic code, and thus encodethe same LMP as that encoded by the nucleotide sequences of the presentinvention. In a further embodiment, the nucleic acid molecule of theinvention encodes a full length protein which is substantiallyhomologous to an amino acid sequence of a polypeptide encoded by an openreading frame specified herein. In one embodiment, the full-lengthnucleic acid or protein or fragment of the nucleic acid or protein isfrom Arabidopsis thaliana, Brassica napus, Glycine max or Oryza sativa.

In addition to the Arabidopsis thaliana, Brassica napus, Glycine max andOryza sativa LMP nucleotide sequences of the present invention, it willbe appreciated by those skilled in the art that DNA sequencepolymorphisms that lead to changes in the amino acid sequences of LMPsmay exist within a population (e.g., the Arabidopsis thaliana, Brassicanapus, Glycine max or Oryza sativa population). Such geneticpolymorphism in the LMP gene may exist among individuals within apopulation due to natural variation. As used herein, the terms “gene”and “recombinant gene” refer to nucleic acid molecules comprising anopen reading frame encoding an LMP, preferably a Arabidopsis thaliana,Brassica napus, Glycine max or Oryza sativa LMP. Such natural variationscan typically result in 1-40% variance in the nucleotide sequence of theLMP gene. Any and all such nucleotide variations and resulting aminoacid polymorphisms in LMP that are the result of natural variation andthat do not alter the functional activity of LMPs are intended to bewithin the scope of the invention.

Nucleic acid molecules corresponding to natural variants andnon-Arabidopsis thaliana, Brassica napus, Glycine max or Oryza sativa,orthologs of the Arabidopsis thaliana, Brassica napus, Glycine max orOryza sativa LMP cDNA of the invention can be isolated based on theirhomology to Arabidopsis thaliana, Brassica napus, Glycine max or Oryzasativa LMP nucleic acid disclosed herein using the Arabidopsis thaliana,Brassica napus, Glycine max or Oryza sativa cDNA, or a portion thereof,as a hybridization probe according to standard hybridization techniquesunder stringent hybridization conditions. As used herein, the term“orthologs” refers to two nucleic acids from different species, but thathave evolved from a common ancestral gene by speciation. Normally,orthologs encode proteins having the same or similar functions.Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 15 nucleotides in length and hybridizes understringent conditions to the nucleic acid molecule comprising anucleotide sequence of the present invention. In other embodiments, thenucleic acid is at least 30, 50, 100, 250, or more nucleotides inlength. As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing, underwhich nucleotide sequences at least 60% homologous to each othertypically remain hybridized to each other. Preferably, the conditionsare such that sequences at least about 65%, more preferably at leastabout 70%, and even more preferably at least about 75% or morehomologous to each other typically remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, John Wiley & Sons,N.Y., 1989: 6.3.1-6.3.6. A preferred, non-limiting example of stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50-65° C. Preferably, an isolated nucleic acidmolecule of the invention that hybridizes under stringent conditions toa sequence of the present invention corresponds to a naturally occurringnucleic acid molecule. As used herein, a “naturally-occurring” nucleicacid molecule refers to an RNA or DNA molecule having a nucleotidesequence that occurs in nature (e.g., encodes a natural protein). In oneembodiment, the nucleic acid encodes a natural Arabidopsis thaliana,Brassica napus, Glycine max, or Oryza sativa LMP.

In addition to naturally-occurring variants of the LMP sequence that mayexist in the population, the skilled artisan will further appreciatethat changes can be introduced by mutation into a nucleotide sequence ofthe present invention, thereby leading to changes in the amino acidsequence of the encoded LMP, without altering the functional ability ofthe LMP. For example, nucleotide substitutions leading to amino acidsubstitutions at “non-essential” amino acid residues can be made in asequence of the present invention. A “non-essential” amino acid residueis a residue that can be altered from the wild-type sequence of one ofthe LMPs without altering the activity of said LMP, whereas an“essential” amino acid residue is required for LMP activity. Other aminoacid residues, however, (e.g., those that are not conserved or onlysemi-conserved in the domain having LMP activity) may not be essentialfor activity, and thus are likely to be amenable to alteration withoutaltering LMP activity.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding LMPs that contain changes in amino acid residues thatare not essential for LMP activity. Such LMPs differ in amino acidsequence from a sequence yet retain at least one of the LMP activitiesdescribed herein. In one embodiment, the isolated nucleic acid moleculecomprises a nucleotide sequence encoding a protein, wherein the proteincomprises an amino acid sequence at least about 50% homologous to anamino acid sequence encoded by a nucleic acid of the present inventionand is capable of participation in the metabolism of compounds necessaryfor the production of seed storage compounds in Arabidopsis thaliana,Brassica napus, Glycine max, Zea mays, Oryza sativa, Triticum aestivum,Hordeum vulgare, Linum usitatissimum, Helianthus anuus, or Beta vulgarisor Physcomitrella patens, or cellular membranes, or has one or moreactivities set forth in Table 15. Preferably, the protein encoded by thenucleic acid molecule is at least about 50-60% homologous to one of thesequences encoded by a specific nucleic acid of the present invention,more preferably at least about 60-70% homologous to one of the sequencesencoded by a specific nucleic acid of the present invention, even morepreferably at least about 70-80%, 80-90%, 90-95% homologous to one ofthe sequences encoded by a specific nucleic acid of the presentinvention, and most preferably at least about 96%, 97%, 98%, or 99%homologous to one of the sequences encoded by a specific nucleic acid ofthe present invention.

To determine the percent homology of two amino acid sequences (e.g., oneof the sequences encoded by a specific nucleic acid of the presentinvention and a mutant form thereof) or of two nucleic acids, thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in the sequence of one protein or nucleic acid for optimalalignment with the other protein or nucleic acid). The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in one sequence(e.g., one of the sequences encoded by a specific nucleic acid of thepresent invention) is occupied by the same amino acid residue ornucleotide as the corresponding position in the other sequence (e.g., amutant form of the sequence selected from the polypeptide encoded by aspecific nucleic acid of the present invention), then the molecules arehomologous at that position (i.e., as used herein amino acid or nucleicacid “homology” is equivalent to amino acid or nucleic acid “identity”).The percent homology between the two sequences is a function of thenumber of identical positions shared by the sequences (i.e., %homology=numbers of identical positions/total numbers of positions×100).

An isolated nucleic acid molecule encoding an LMP homologous to aprotein sequence encoded by a nucleic acid of the present invention canbe created by introducing one or more nucleotide substitutions,additions or deletions into a nucleotide sequence of the presentinvention, such that one or more amino acid substitutions, additions ordeletions are introduced into the encoded protein. Mutations can beintroduced into one of the sequences of the present invention bystandard techniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions are madeat one or more predicted non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine), and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted non-essential amino acid residue in an LMP is preferablyreplaced with another amino acid residue from the same side chainfamily. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of an LMP coding sequence, such asby saturation mutagenesis, and the resultant mutants can be screened foran LMP activity described herein to identify mutants that retain LMPactivity. Following mutagenesis of one of the sequences of the presentinvention, the encoded protein can be expressed recombinantly and theactivity of the protein can be determined using, for example, assaysdescribed herein (see Examples 11-13 of the Exemplification).

LMPs are preferably produced by recombinant DNA techniques. For example,a nucleic acid molecule encoding the protein is cloned into anexpression vector (as described above), the expression vector isintroduced into a host cell (as described herein), and the LMP isexpressed in the host cell. The LMP can then be isolated from the cellsby an appropriate purification scheme using standard proteinpurification techniques. Alternative to recombinant expression, an LMPor peptide thereof can be synthesized chemically using standard peptidesynthesis techniques. Moreover, native LMP can be isolated from cells,for example using an anti-LMP antibody, which can be produced bystandard techniques utilizing an LMP or fragment thereof of thisinvention.

The invention also provides LMP chimeric or fusion proteins. As usedherein, an LMP “chimeric protein” or “fusion protein” comprises an LMPpolypeptide operatively linked to a non-LMP polypeptide. An “LMPpolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to an LMP, whereas a “non-LMP polypeptide” refers to apolypeptide having an amino acid sequence corresponding to a proteinwhich is not substantially homologous to the LMP, e.g., a protein thatis different from the LMP, and which is derived from the same or adifferent organism. Within the fusion protein, the term “operativelylinked” is intended to indicate that the LMP polypeptide and the non-LMPpolypeptide are fused to each other so that both sequences fulfill theproposed function attributed to the sequence used. The non-LMPpolypeptide can be fused to the N-terminus or C-terminus of the LMPpolypeptide. For example, in one embodiment, the fusion protein is aGST-LMP (glutathione S-transferase) fusion protein in which the LMPsequences are fused to the C-terminus of the GST sequences. Such fusionproteins can facilitate the purification of recombinant LMPs.

In another embodiment, the fusion protein is an LMP containing aheterologous signal sequence at its N-terminus. In certain host cells(e.g., mammalian host cells), expression and/or secretion of an LMP canbe increased through use of a heterologous signal sequence.

Preferably, an LMP chimeric or fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primersthat give rise to complementary overhangs between two consecutive genefragments, which can subsequently be annealed and reamplified togenerate a chimeric gene sequence (see, for example, Current Protocolsin Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). AnLMP-encoding nucleic acid can be cloned into such an expression vectorsuch that the fusion moiety is linked in-frame to the LMP.

In addition to the nucleic acid molecules encoding LMPs described above,another aspect of the invention pertains to isolated nucleic acidmolecules that are antisense thereto. An “antisense” nucleic acidcomprises a nucleotide sequence that is complementary to a “sense”nucleic acid encoding a protein, e.g., complementary to the codingstrand of a double-stranded cDNA molecule or complementary to an mRNAsequence. Accordingly, an antisense nucleic acid can hydrogen bond to asense nucleic acid. The antisense nucleic acid can be complementary toan entire LMP coding strand, or to only a portion thereof. In oneembodiment, an antisense nucleic acid molecule is antisense to a “codingregion” of the coding strand of a nucleotide sequence encoding an LMP.The term “coding region” refers to the region of the nucleotide sequencecomprising codons that are translated into amino acid residues (e.g.,the entire coding region of pk331OS37109650 comprises nucleotides82-1704). In another embodiment, the antisense nucleic acid molecule isantisense to a “noncoding region” of the coding strand of a nucleotidesequence encoding LMP. The term “noncoding region” refers to 5′ and 3′sequences that flank the coding region that are not translated intoamino acids (i.e., also referred to as 5′ and 3′ untranslated regions).

Given the coding strand sequences encoding LMP disclosed herein (e.g.,the specific sequences set forth elsewhere herein), antisense nucleicacids of the invention can be designed according to the rules of Watsonand Crick base pairing. The antisense nucleic acid molecule can becomplementary to the entire coding region of LMP mRNA, but morepreferably is an oligonucleotide that is antisense to only a portion ofthe coding or noncoding region of LMP mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of LMP mRNA. An antisense oligonucleotide can be,for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotidesin length. An antisense or sense nucleic acid of the invention can beconstructed using chemical synthesis and enzymatic ligation reactionsusing procedures known in the art. For example, an antisense nucleicacid (e.g., an antisense oligonucleotide) can be chemically synthesizedusing naturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used. Examples of modifiednucleotides which can be used to generate the antisense nucleic acidinclude 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylamino-methyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydro-uracil,beta-D-galactosylqueosine, inosine, N-6-isopentenyladenine,1-methyl-guanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methyl-cytosine, N-6-adenine,7-methylguanine, 5-methyl-aminomethyluracil,5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyl-uracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diamino-purine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector, into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

In another variation of the antisense technology, a double-strand.interfering RNA construct can be used to cause a down-regulation of theLMP mRNA level and LMP activity in transgenic plants. This requirestransforming the plants with a chimeric construct containing a portionof the LMP sequence in the sense orientation fused to the antisensesequence of the same portion of the LMP sequence. A DNA linker region ofvariable length can be used to separate the sense and antisensefragments of LMP sequences in the construct.

The antisense nucleic acid molecules of the invention are typicallyadministered to a cell or generated in situ, such that they hybridizewith, or bind to, cellular mRNA and/or genomic DNA encoding an LMP tothereby inhibit expression of the protein, e.g., by inhibitingtranscription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule, which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. The antisense molecule can be modified such that itspecifically binds to a receptor or an antigen expressed on a selectedcell surface, e.g., by linking the antisense nucleic acid molecule to apeptide or an antibody, which binds to a cell surface receptor orantigen. The antisense nucleic acid molecule can also be delivered tocells using the vectors described herein. To achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs, in which the antisense nucleic acid molecule is placed underthe control of a strong prokaryotic, viral, or eukaryotic includingplant promoters, are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an anomeric nucleic acid molecule. An anomeric nucleic acidmolecule forms specific double-stranded hybrids with complementary RNA,in which, contrary to the usual units, the strands run parallel to eachother (Gaultier et al. 1987, Nucleic Acids Res. 15:6625-6641). Theantisense nucleic acid molecule can also comprise a2′-o-methyl-ribonucleotide (Inoue et al. 1987, Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. 1987, FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity, which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes (described in Haselhoff & Gerlach1988, Nature 334:585-591)) can be used to catalytically cleave LMP mRNAtranscripts to thereby inhibit translation of LMP mRNA. A ribozymehaving specificity for an LMP-encoding nucleic acid can be designedbased upon the nucleotide sequence of an LMP cDNA disclosed herein(i.e., Bn01, below) or on the basis of a heterologous sequence to beisolated according to methods taught in this invention. For example, aderivative of a Tetrahymena L-19 IVS RNA can be constructed, in whichthe nucleotide sequence of the active site is complementary to thenucleotide sequence to be cleaved in an LMP-encoding mRNA (see, e.g.,Cech et al., U.S. Pat. No. 4,987,071 and Cech et al., U.S. Pat. No.5,116,742). Alternatively, LMP mRNA can be used to select a catalyticRNA having a specific ribonuclease activity from a pool of RNA molecules(see, e.g., Bartel, D. & Szostak J. W. 1993, Science 261:1411-1418).

Alternatively, LMP gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of an LMPnucleotide sequence (e.g., an LMP promoter and/or enhancers) to formtriple helical structures that prevent transcription of an LMP gene intarget cells (See generally, Helene C. 1991, Anticancer Drug Des.6:569-84; Helene C. et al. 1992, Ann. N.Y. Acad. Sci. 660:27-36; andMaher, L. J. 1992, Bioassays 14:807-15).

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding an LMP (or aportion thereof). As used herein, the term “vector” refers to a nucleicacid molecule capable of transporting another nucleic acid, to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular, double-stranded DNA loop, into which additional DNA segmentscan be ligated. Another type of vector is a viral vector, whereinadditional DNA segments can be ligated into the viral genome. Certainvectors are capable of autonomous replication in a host cell, into whichthey are introduced (e.g., bacterial vectors having a bacterial originof replication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes, to which they are operatively linked.Such vectors are referred to herein as “expression vectors.” In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerthat allows for expression of the nucleotide sequence and both sequencesare fused to each other so that each fulfills its proposed function(e.g., in an in vitro transcription/translation system or in a host cellwhen the vector is introduced into the host cell). The term “regulatorysequence” is intended to include promoters, enhancers, and otherexpression-control elements (e.g., polyadenylation signals). Suchregulatory sequences are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) or see: Gruber and Crosby, in: Methods in PlantMolecular Biology and Biotechnology, CRC Press, Boca Raton, Fla., eds.:Glick & Thompson, Chapter 7, 89-108 including the references therein.Regulatory sequences include those that direct constitutive expressionof a nucleotide sequence in many types of host cell and those thatdirect expression of the nucleotide sequence only in certain host cellsor under certain conditions. It will be appreciated by those skilled inthe art that the design of the expression vector can depend on suchfactors as the choice of the host cell to be transformed, the level ofexpression of protein desired, etc. The expression vectors of theinvention can be introduced into host cells to thereby produce proteinsor peptides, including fusion proteins or peptides, encoded by nucleicacids as described herein (e.g., LMPs, mutant forms of LMPs, fusionproteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of LMPs in prokaryotic or eukaryotic cells. For example, LMPgenes can be expressed in bacterial cells, insect cells (usingbaculovirus expression vectors), yeast and other fungal cells (seeRomanos M. A. et al. 1992, Foreign gene expression in yeast: a review,Yeast 8:423-488; van den Hondel, C. A. M. J. J. et al. 1991,Heterologous gene expression in filamentous fungi, in: More GeneManipulations in Fungi, Bennet & Lasure, eds., p. 396-428: AcademicPress: an Diego; and van den Hondel & Punt 1991, Gene transfer systemsand vector development for filamentous fungi, in: Applied MolecularGenetics of Fungi, Peberdy et al., eds., p. 1-28, Cambridge UniversityPress: Cambridge), algae (Falciatore et al. 1999, Marine Biotechnology1:239-251), ciliates of the types: Holotrichia, Peritrichia,Spirotrichia, Suctoria, Tetrahymena, Paramecium, Colpidium, Glaucoma,Platyophrya, Potomacus, Pseudocohnilembus, Euplotes, Engelmaniella, andStylonychia, especially of the genus Stylonychia lemnae with vectorsfollowing a transformation method as described in WO 98/01572 andmulticellular plant cells (see Schmidt & Willmitzer 1988, Highefficiency Agrobacterium tumefaciens-mediated transformation ofArabidopsis thaliana leaf and cotyledon plants, Plant Cell Rep.:583-586); Plant Molecular Biology and Biotechnology, C Press, BocaRaton, Fla., chapter 6/7, S.71-119 (1993); White, Jenes et al.,Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineeringand Utilization, eds.: Kung and Wu, Academic Press 1993, 128-43;Potrykus 1991, Annu. Rev. Plant Physiol. Plant Mol. Biol. 42:205-225(and references cited therein) or mammalian cells. Suitable host cellsare discussed further in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. 1990). Alternatively,the recombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression of proteins in prokaryotes is most often carried out withvectors containing constitutive or inducible promoters directing theexpression of either fusion or non-fusion proteins. Fusion vectors add anumber of amino acids to a protein encoded therein, usually to the aminoterminus of the recombinant protein but also to the C-terminus or fusedwithin suitable regions in the proteins. Such fusion vectors typicallyserve one or more of the following purposes: 1) to increase expressionof recombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin, and enterokinase.

Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith & Johnson 1988, Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.), which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein. In one embodiment,the coding sequence of the LMP is cloned into a pGEX expression vectorto create a vector encoding a fusion protein comprising, from theN-terminus to the C-terminus, GST-thrombin cleavage site-X protein. Thefusion protein can be purified by affinity chromatography usingglutathione-agarose resin. Recombinant LMP unfused to GST can berecovered by cleavage of the fusion protein with thrombin.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al. 1988, Gene 69:301-315) and pET 11d (Studieret al. 1990, Gene Expression Technology Methods in Enzymology 185,Academic Press, San Diego, Calif. 60-89). Target gene expression fromthe pTrc vector relies on host RNA polymerase transcription from ahybrid trp-lac fusion promoter. Target gene expression from the pET 11dvector relies on transcription from a T7 gn10-lac fusion promotermediated by a coexpressed viral RNA polymerase (T7 gn1). This viralpolymerase is supplied by host strains BL21 (DE3) or HMS174 (DE3) from aresident prophage harboring a T7 gn1 gene under the transcriptionalcontrol of the lacUV 5 promoter.

One strategy to maximize recombinant protein expression is to expressthe protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman S. 1990, GeneExpression Technology: Methods in Enzymology 185:119-128, AcademicPress, San Diego, Calif.). Another strategy is to alter the nucleic acidsequence of the nucleic acid to be inserted into an expression vector sothat the individual codons for each amino acid are those preferentiallyutilized in the bacterium chosen for expression (Wada et al. 1992,Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acidsequences of the invention can be carried out by standard DNA synthesistechniques.

In another embodiment, the LMP expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari et al. 1987, Embo J. 6:229-234), pMFa (Kurjan& Herskowitz 1982, Cell 30:933-943), pJRY88 (Schultz et al. 1987, Gene54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.).Vectors and methods for the construction of vectors appropriate for usein other fungi, such as the filamentous fungi, include those detailedin: van den Hondel & Punt 1991, “Gene transfer systems and vectordevelopment for filamentous fungi, in: Applied Molecular Genetics ofFungi, Peberdy et al., eds., p. 1-28, Cambridge University Press:Cambridge.

Alternatively, the LMPs of the invention can be expressed in insectcells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. 1983, Mol. Cell. Biol.3:2156-2165) and the pVL series (Lucklow & Summers 1989, Virology170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed 1987, Nature 329:840)and pMT2PC (Kaufman et al. 1987, EMBO J. 6:187-195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus, andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook,Fritsh and Maniatis, Molecular Cloning: A Laboratory Manual. 2nd, ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

In another embodiment, the LMPs of the invention may be expressed inunicellular plant cells (such as algae, see Falciatore et al. (1999,Marine Biotechnology 1:239-251 and references therein) and plant cellsfrom higher plants (e.g., the spermatophytes, such as crop plants).Examples of plant expression vectors include those detailed in: Becker,Kemper, Schell and Masterson (1992, “New plant binary vectors withselectable markers located proximal to the left border”, Plant Mol.Biol. 20:1195-1197) and Bevan (1984, “Binary Agrobacterium vectors forplant transformation, Nucleic Acids Res. 12:8711-8721; Vectors for GeneTransfer in Higher Plants; in: Transgenic Plants, Vol. 1, Engineeringand Utilization, eds.: Kung und R. Wu, Academic Press, 1993, S. 15-38).

A plant expression cassette preferably contains regulatory sequencescapable to drive gene expression in plant cells, and which are operablylinked so that each sequence can fulfill its function such astermination of transcription, including polyadenylation signals.Preferred polyadenylation signals are those originating fromAgrobacterium tumefaciens t-DNA such as the gene 3 known as octopinesynthase of the Ti-plasmid pTiACH5 (Gielen et al. 1984, EMBO J. 3:835)or functional equivalents thereof but also all other terminatorsfunctionally active in plants are suitable.

As plant gene expression is very often not limited on transcriptionallevels a plant expression cassette preferably contains other operablylinked sequences like translational enhancers such as theoverdrive-sequence containing the 5′-untranslated leader sequence fromtobacco mosaic virus enhancing the protein per RNA ratio (Gallie et al.1987, Nucleic Acids Res. 15:8693-8711).

Plant gene expression has to be operably linked to an appropriatepromoter conferring gene expression in a timely, cell or tissue specificmanner. Preferred are promoters driving constitutive expression (Benfeyet al. 1989, EMBO J. 8:2195-2202) like those derived from plant viruseslike the 35S CAMV (Franck et al. 1980, Cell 21:285-294), the 19S CaMV(see also U.S. Pat. No. 5,352,605 and WO 84/02913) or plant promoterslike those from Rubisco small subunit described in U.S. Pat. No.4,962,028. Even more preferred are seed-specific promoters drivingexpression of LMP proteins during all or selected stages of seeddevelopment. Seed-specific plant promoters are known to those ofordinary skill in the art and are identified and characterized usingseed-specific mRNA libraries and expression profiling techniques.Seed-specific promoters include the napin-gene promoter from rapeseed(U.S. Pat. No. 5,608,152), the USP-promoter from Vicia faba (Baeumleinet al. 1991, Mol. Gen. Genetics 225:459-67), the oleosin-promoter fromArabidopsis (WO 98/45461), the phaseolin-promoter from Phaseolusvulgaris (U.S. Pat. No. 5,504,200), the Bce-4-promoter from Brassica(WO9113980) or the legumin B4 promoter (LeB4; Baeumlein et al. 1992,Plant J. 2:233-239) as well as promoters conferring seed specificexpression in monocot plants like maize, barley, wheat, rye, rice etc.Suitable promoters to note are the Ipt2 or Ipt1-gene promoter frombarley (WO 95/15389 and WO 95/23230) or those described in WO 99/16890(promoters from the barley hordein-gene, the rice glutelin gene, therice oryzin gene, the rice prolamin gene, the wheat gliadin gene, wheatglutelin gene, the maize zein gene, the oat glutelin gene, the Sorghumkasirin-gene, and the rye secalin gene).

Plant gene expression can also be facilitated via an inducible promoter(for a review see Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol.48:89-108). Chemically inducible promoters are especially suitable ifgene expression is desired in a time specific manner. Examples for suchpromoters are a salicylic acid inducible promoter (WO 95/19443), atetracycline inducible promoter (Gatz et al. 1992, Plant J. 2:397-404),and an ethanol inducible promoter (WO 93/21334).

Promoters responding to biotic or abiotic stress conditions are alsosuitable promoters, such as the pathogen inducible PRP1-gene promoter(Ward et al., 1993, Plant. Mol. Biol. 22:361-366), the heat induciblehsp80-promoter from tomato (U.S. Pat. No. 5,187,267), cold induciblealpha-amylase promoter from potato (WO 96/12814), or the wound-induciblepinII-promoter (EP 375091).

Other preferred sequences for use in plant gene expression cassettes aretargeting-sequences necessary to direct the gene-product in itsappropriate cell compartment (for review see Kermode 1996, Crit. Rev.Plant Sci. 15:285-423 and references cited therein) such as the vacuole,the nucleus, all types of plastids like amyloplasts, chloroplasts,chromoplasts, the extracellular space, mitochondria, the endoplasmicreticulum, oil bodies, peroxisomes and other compartments of plantcells. Also especially suited are promoters that confer plastid-specificgene expression, as plastids are the compartment where precursors andsome end products of lipid biosynthesis are synthesized. Suitablepromoters such as the viral RNA-polymerase promoter are described in WO95/16783 and WO 97/06250 and the clpP-promoter from Arabidopsisdescribed in WO 99/46394.

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner that allows forexpression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to LMP mRNA. Regulatory sequences operatively linkedto a nucleic acid cloned in the antisense orientation can be chosenwhich direct the continuous expression of the antisense RNA molecule ina variety of cell types, for instance viral promoters and/or enhancers,or regulatory sequences can be chosen that direct constitutive, tissuespecific or cell type specific expression of antisense RNA. Theantisense expression vector can be in the form of a recombinant plasmid,phagemid or attenuated virus, in which antisense nucleic acids areproduced under the control of a high efficiency regulatory region, theactivity of which can be determined by the cell type into which thevector is introduced. For a discussion of the regulation of geneexpression using antisense genes, see Weintraub et al. (1986, AntisenseRNA as a molecular tool for genetic analysis, Reviews—Trends inGenetics, Vol. 1) and Mol et al. (1990, FEBS Lett. 268:427-430).

Another aspect of the invention pertains to host cells, into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is to be understood that such terms refer not only to theparticular subject cell but also to the progeny or potential progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein. A host cell can beany prokaryotic or eukaryotic cell. For example, an LMP can be expressedin bacterial cells, insect cells, fungal cells, mammalian cells (such asChinese hamster ovary cells (CHO) or COS cells), algae, ciliates, orplant cells. Other suitable host cells are known to those skilled in theart.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection,” “conjugation” and“transduction” are intended to refer to a variety of art-recognizedtechniques for introducing foreign nucleic acid (e.g., DNA) into a hostcell, including calcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection, natural competence,chemical-mediated transfer, or electroporation. Suitable methods fortransforming or transfecting host cells including plant cells can befound in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.) and other laboratory manuals such asMethods in Molecular Biology 1995, Vol. 44, Agrobacterium protocols, ed:Gartland and Davey, Humana Press, Totowa, N.J.

For stable transfection of mammalian and plant cells, it is known that,depending upon the expression vector and transfection technique used,only a small fraction of cells may integrate the foreign DNA into theirgenome. In order to identify and select these integrants, a gene thatencodes a selectable marker (e.g., resistance to antibiotics) isgenerally introduced into the host cells along with the gene ofinterest. Preferred selectable markers include those that conferresistance to drugs, such as G418, hygromycin, kanamycin, andmethotrexate, or in plants that confer resistance towards an herbicide,such as glyphosate or glufosinate. A nucleic acid encoding a selectablemarker can be introduced into a host cell on the same vector as thatencoding an LMP or can be introduced on a separate vector. Cells stablytransfected with the introduced nucleic acid can be identified by, forexample, drug selection (e.g., cells that have incorporated theselectable marker gene will survive, while the other cells die).

To create a homologous recombinant microorganism, a vector is preparedthat contains at least a portion of an LMP gene, into which a deletion,addition, or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the LMP gene. Preferably, this LMP gene is anArabidopsis thaliana, Brassica napus or Glycine max LMP gene, but it canbe a homologue from a related plant or even from a mammalian, yeast, orinsect source. In a preferred embodiment, the vector is designed suchthat, upon homologous recombination, the endogenous LMP gene isfunctionally disrupted (i.e., no longer encodes a functional protein;also referred to as a knock-out vector). Alternatively, the vector canbe designed such that, upon homologous recombination, the endogenous LMPgene is mutated or otherwise altered but still encodes functionalprotein (e.g., the upstream regulatory region can be altered to therebyalter the expression of the endogenous LMP). To create a point mutationvia homologous recombination, DNA-RNA hybrids can be used in a techniqueknown as chimeraplasty (Cole-Strauss et al. 1999, Nucleic Acids Res.27:1323-1330 and Kmiec 1999, American Scientist 87:240-247). Homologousrecombination procedures in Arabidopsis thaliana or other crops are alsowell known in the art and are contemplated for use herein.

In a homologous recombination vector, the altered portion of the LMPgene is flanked at its 5′ and 3′ ends by additional nucleic acid of theLMP gene to allow for homologous recombination to occur between theexogenous LMP gene carried by the vector and an endogenous LMP gene in amicroorganism or plant. The additional flanking LMP nucleic acid is ofsufficient length for successful homologous recombination with theendogenous gene. Typically, several hundreds of base pairs up tokilobases of flanking DNA (both at the 5′ and 3′ ends) are included inthe vector (see e.g., Thomas & Capecchi 1987, Cell 51:503, for adescription of homologous recombination vectors). The vector isintroduced into a microorganism or plant cell (e.g., viapolyethyleneglycol mediated DNA). Cells in which the introduced LMP genehas homologously recombined with the endogenous LMP gene are selectedusing art-known techniques.

In another embodiment, recombinant microorganisms can be produced thatcontain selected systems that allow for regulated expression of theintroduced gene. For example, inclusion of an LMP gene on a vectorplacing it under control of the lac operon permits expression of the LMPgene only in the presence of IPTG. Such regulatory systems are wellknown in the art.

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture can be used to produce (i.e., express) an LMP.Accordingly, the invention further provides methods for producing LMPsusing the host cells of the invention. In one embodiment, the methodcomprises culturing a host cell of the invention (into which arecombinant expression vector encoding an LMP has been introduced, orwhich contains a wild-type or altered LMP gene in its genome) in asuitable medium until LMP is produced. In another embodiment, the methodfurther comprises isolating LMPs from the medium or the host cell.

Another aspect of the invention pertains to isolated LMPs, andbiologically active portions thereof. An “isolated” or “purified”protein or biologically active portion thereof is substantially free ofcellular material when produced by recombinant DNA techniques, orchemical precursors or other chemicals when chemically synthesized. Thelanguage “substantially free of cellular material” includes preparationsof LMP, in which the protein is separated from cellular components ofthe cells, in which it is naturally or recombinantly produced. In oneembodiment, the language “substantially free of cellular material”includes preparations of LMP having less than about 30% (by dry weight)of non-LMP (also referred to herein as a “contaminating protein”), morepreferably less than about 20% of non-LMP, still more preferably lessthan about 10% of non-LMP, and most preferably less than about 5%non-LMP. When the LMP or biologically active portion thereof isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the protein preparation. The language “substantiallyfree of chemical precursors or other chemicals” includes preparations ofLMP in which the protein is separated from chemical precursors or otherchemicals that are involved in the synthesis of the protein. In oneembodiment, the language “substantially free of chemical precursors orother chemicals” includes preparations of LMP having less than about 30%(by dry weight) of chemical precursors or non-LMP chemicals, morepreferably less than about 20% chemical precursors or non-LMP chemicals,still more preferably less than about 10% chemical precursors or non-LMPchemicals, and most preferably less than about 5% chemical precursors ornon-LMP chemicals. In preferred embodiments, isolated proteins orbiologically active portions thereof lack contaminating proteins fromthe same organism from which the LMP is derived. Typically, suchproteins are produced by recombinant expression of, for example, anArabidopsis thaliana LMP in other plants than Arabidopsis thaliana orthe moss Physcomitrella patens or microorganisms, algae or fungi.

An isolated LMP or a portion thereof of the invention can participate inthe metabolism of compounds necessary for the production of seed storagecompounds in Arabidopsis thaliana, Brassica napus, Glycine max, Zeamays, Oryza sativa, Linum usitatissimum, Hordeum vulgare, Triticumaestivum, Helianthus anuus, or Beta vulgaris or Physcomitrella patens orof cellular membranes, or has one or more of the activities set forth inTable 15. In preferred embodiments, the protein or portion thereofcomprises an amino acid sequence which is sufficiently homologous to anamino acid sequence encoded by a nucleic acid of the present invention,such that the protein or portion thereof maintains the ability toparticipate in the metabolism of compounds necessary for theconstruction of cellular membranes in Arabidopsis thaliana, Brassicanapus, Glycine max, Zea mays, Oryza sativa, Linum usitatissimum, Hordeumvulgare, Triticum aestivum, Helianthus anuus, Beta vulgaris orPhyscomitrella patens, or in the transport of molecules across thesemembranes. The portion of the protein is preferably a biologicallyactive portion as described herein. In another preferred embodiment, anLMP of the invention has an amino acid sequence encoded by a nucleicacid of the present invention. In yet another preferred embodiment, theLMP has an amino acid sequence, which is encoded by a nucleotidesequence, which hybridizes, e.g., hybridizes under stringent conditions,to a nucleotide sequence of the present invention. In still anotherpreferred embodiment, the LMP has an amino acid sequence, which isencoded by a nucleotide sequence that is at least about 50-60%,preferably at least about 60-70%, more preferably at least about 70-80%,80-90%, 90-95%, and even more preferably at least about 96%, 97%, 98%,99% or more homologous to one of the amino acid sequences encoded by anucleic acid of the present invention. The preferred LMPs of the presentinvention also preferably possess at least one of the LMP activitiesdescribed herein. For example, a preferred LMP of the present inventionincludes an amino acid sequence encoded by a nucleotide sequence whichhybridizes, e.g., hybridizes under stringent conditions, to a nucleotidesequence of the present invention and which can participate in themetabolism of compounds necessary for the construction of cellularmembranes in Arabidopsis thaliana, Brassica napus, Glycine max, Zeamays, Oryza sativa, Linum usitatissimum, Hordeum vulgare, Triticumaestivum, Helianthus anuus, Beta vulgaris, or Physcomitrella patens, orin the transport of molecules across these membranes, or which has oneor more of the activities set forth in Table 15.

In other embodiments, the LMP is substantially homologous to an aminoacid sequence encoded by a nucleic acid of the present invention andretains the functional activity of the protein of one of the sequencesencoded by a nucleic acid of the present invention yet differs in aminoacid sequence due to natural variation or mutagenesis, as described indetail above. Accordingly, in another embodiment, the LMP is a proteinwhich comprises an amino acid sequence which is at least about 50-60%,preferably at least about 60-70%, and more preferably at least about70-80, 80-90, 90-95%, and most preferably at least about 96%, 97%, 98%,99%, or more homologous to an entire amino acid sequence and which hasat least one of the LMP activities described herein. In anotherembodiment, the invention pertains to a full Arabidopsis thaliana,Brassica napus, Glycine max or Oryza sativa protein which issubstantially homologous to an entire amino acid sequence encoded by anucleic acid of the present invention.

Dominant negative mutations or trans-dominant suppression can be used toreduce the activity of an LMP in transgenics seeds in order to changethe levels of seed storage compounds. To achieve this a mutation thatabolishes the activity of the LMP is created and the inactivenon-functional LMP gene is overexpressed in the transgenic plant. Theinactive trans-dominant LMP protein competes with the active endogenousLMP protein for substrate or interactions with other proteins anddilutes out the activity of the active LMP. In this way the biologicalactivity of the LMP is reduced without actually modifying the expressionof the endogenous LMP gene. This strategy was used by Pontier et al tomodulate the activity of plant transcription factors (Pontier D, Miao ZH, Lam E, Plant J 2001 Sep. 27(6): 529-38, Trans-dominant suppression ofplant TGA factors reveals their negative and positive roles in plantdefense responses).

Homologues of the LMP can be generated by mutagenesis, e.g., discretepoint mutation or truncation of the LMP. As used herein, the term“homologue” refers to a variant form of the LMP that acts as an agonistor antagonist of the activity of the LMP. An agonist of the LMP canretain substantially the same, or a subset, of the biological activitiesof the LMP. An antagonist of the LMP can inhibit one or more of theactivities of the naturally occurring form of the LMP, by, for example,competitively binding to a downstream or upstream member of the cellmembrane component metabolic cascade which includes the LMP, or bybinding to an LMP which mediates transport of compounds across suchmembranes, thereby preventing translocation from taking place.

In an alternative embodiment, homologues of the LMP can be identified byscreening combinatorial libraries of mutants, e.g., truncation mutants,of the LMP for LMP agonist or antagonist activity. In one embodiment, avariegated library of LMP variants is generated by combinatorialmutagenesis at the nucleic acid level and is encoded by a variegatedgene library. A variegated library of LMP variants can be produced by,for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential LMP sequences is expressible as individual polypeptides, oralternatively, as a set of larger fusion proteins (e.g., for phagedisplay) containing the set of LMP sequences therein. There are avariety of methods that can be used to produce libraries of potentialLMP homologues from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be performed in an automaticDNA synthesizer, and the synthetic gene then ligated into an appropriateexpression vector. Use of a degenerate set of genes allows for theprovision, in one mixture, of all of the sequences encoding the desiredset of potential LMP sequences. Methods for synthesizing degenerateoligonucleotides are known in the art (see, e.g., Narang 1983,Tetrahedron 39:3; Itakura et al. 1984, Annu. Rev. Biochem. 53:323;Itakura et al. 1984, Science 198:1056; Ike et al. 1983, Nucleic AcidsRes. 11:477).

In addition, libraries of fragments of the LMP coding sequences can beused to generate a variegated population of LMP fragments for screeningand subsequent selection of homologues of an LMP. In one embodiment, alibrary of coding sequence fragments can be generated by treating adouble-stranded PCR fragment of an LMP coding sequence with a nucleaseunder conditions, wherein nicking occurs only about once per molecule,denaturing the double stranded DNA, renaturing the DNA to form doublestranded DNA, which can include sense/antisense pairs from differentnicked products, removing single stranded portions from reformedduplexes by treatment with S1 nuclease, and ligating the resultingfragment library into an expression vector. By this method, anexpression library can be derived, which encodes N-terminal, C-terminaland internal fragments of various sizes of the LMP.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of LMP homologues. The mostwidely used techniques, which are amenable to high through-put analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions, in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a newtechnique that enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify LMP homologues (Arkin & Yourvan 1992, Proc. Natl. Acad. Sci.USA 89:7811-7815; Delgrave et al. 1993, Protein Engineering 6:327-331).

In another embodiment, cell based assays can be exploited to analyze avariegated LMP library, using methods well known in the art.

The nucleic acid molecules, proteins, protein homologues, fusionproteins, primers, vectors, and host cells described herein can be usedin one or more of the following methods: identification of Arabidopsisthaliana, Brassica napus, Glycine max, Zea mays, Oryza sativa, Linumusitatissimum, Hordeum vulgare, Triticum aestivum, Helianthus anuus,Beta vulgaris or Physcomitrella patens and related organisms; mapping ofgenomes of organisms related to Arabidopsis thaliana, Brassica napus,Glycine max, Zea mays, Oryza sativa, Linum usitatissimum, Hordeumvulgare, Triticum aestivum, Helianthus anuus, Beta vulgaris orPhyscomitrella patens; identification and localization of Arabidopsisthaliana, Brassica napus, Glycine max, Zea mays, Oryza sativa, Linumusitatissimum, Hordeum vulgare, Triticum aestivum, Helianthus anuus,Beta vulgaris or Physcomitrella patens sequences of interest;evolutionary studies; determination of LMP regions required forfunction; modulation of an LMP activity; modulation of the metabolism ofone or more cell functions; modulation of the transmembrane transport ofone or more compounds; and modulation of seed storage compoundaccumulation.

The plant Arabidopsis thaliana represents one member of higher (or seed)plants. It is related to other plants such as Brassica napus, Glycinemax, Zea mays, Linum usitatissimum, Hordeum vulgare, Oryza sativa,Helianthus anuus, Beta vulgaris or Triticum aestivum which require lightto drive photosynthesis and growth. Plants like Arabidopsis thaliana,Brassica napus, Glycine max, Zea mays, Linum usitatissimum, Hordeumvulgare, Oryza sativa, Helianthus anuus, Beta vulgaris, Triticumaestivum or the moss Physcomitrella patens share a high degree ofhomology on the DNA sequence and polypeptide level, allowing the use ofheterologous screening of DNA molecules with probes evolving from otherplants or organisms, thus enabling the derivation of a consensussequence suitable for heterologous screening or functional annotationand prediction of gene functions in third species. The ability toidentify such functions can therefore have significant relevance, e.g.,prediction of substrate specificity of enzymes. Further, these nucleicacid molecules may serve as reference points for the mapping ofArabidopsis genomes, or of genomes of related organisms.

The LMP nucleic acid molecules of the invention have a variety of uses.First, the nucleic acid and protein molecules of the invention may serveas markers for specific regions of the genome. This has utility not onlyin the mapping of the genome, but also for functional studies ofArabidopsis thaliana, Brassica napus, Glycine max, Zea mays, Oryzasativa, Linum usitatissimum, Hordeum vulgare, Triticum aestivum,Helianthus anuus, Beta vulgaris or Physcomitrella patens proteins. Forexample, to identify the region of the genome to which a particularArabidopsis thaliana, Brassica napus, Glycine max, Zea mays, Linumusitatissimum, Hordeum vulgare, Oryza sativa, Triticum aestivum,Helianthus anuus, Beta vulgaris or Physcomitrella patens DNA-bindingprotein binds, the Arabidopsis thaliana, Brassica napus, Glycine max,Zea mays, Linum usitatissimum, Hordeum vulgare, Oryza sativa, Triticumaestivum, Helianthus anuus, Beta vulgaris or Physcomitrella patensgenome could be digested, and the fragments incubated with theDNA-binding protein. Those that bind the protein may be additionallyprobed with the nucleic acid molecules of the invention, preferably withreadily detectable labels; binding of such a nucleic acid molecule tothe genome fragment enables the localization of the fragment to thegenome map of Arabidopsis thaliana, Brassica napus, Glycine max, Zeamays, Linum usitatissimum, Hordeum vulgare, Oryza sativa, Triticumaestivum, Helianthus anuus, Beta vulgaris or Physcomitrella patens, and,when performed multiple times with different enzymes, facilitates arapid determination of the nucleic acid sequence to which the proteinbinds. Further, the nucleic acid molecules of the invention may besufficiently homologous to the sequences of related species such thatthese nucleic acid molecules may serve as markers for the constructionof a genomic map in related plants.

The LMP nucleic acid molecules of the invention are also useful forevolutionary and protein structural studies. The metabolic and transportprocesses, in which the molecules of the invention participate areutilized by a wide variety of prokaryotic and eukaryotic cells; bycomparing the sequences of the nucleic acid molecules of the presentinvention to those encoding similar enzymes from other organisms, theevolutionary relatedness of the organisms can be assessed. Similarly,such a comparison permits an assessment, of which regions of thesequence are conserved and which are not, which may aid in determiningthose regions of the protein, which are essential for the functioning ofthe enzyme. This type of determination is of value for proteinengineering studies and may give an indication of what the protein cantolerate in terms of mutagenesis without losing function.

Manipulation of the LMP nucleic acid molecules of the invention mayresult in the production of LMPs having functional differences from thewild-type LMPs. These proteins may be improved in efficiency oractivity, may be present in greater numbers in the cell than is usual,or may be decreased in efficiency or activity.

There are a number of mechanisms by which the alteration of an LMP ofthe invention may directly affect the accumulation and/or composition ofseed storage compounds. In the case of plants expressing LMPs, increasedtransport can lead to altered accumulation of compounds and/or solutepartitioning within the plant tissue and organs which ultimately couldbe used to affect the accumulation of one or more seed storage compoundsduring seed development. An example is provided by Mitsukawa et al.(1997, Proc. Natl. Acad. Sci. USA 94:7098-7102), where overexpression ofan Arabidopsis high-affinity phosphate transporter gene in tobaccocultured cells enhanced cell growth under phosphate-limited conditions.Phosphate availability also affects significantly the production ofsugars and metabolic intermediates (Hurry et al. 2000, Plant J.24:383-396) and the lipid composition in leaves and roots (Härtel et al.2000, Proc. Natl. Acad. Sci. USA 97:10649-10654). Likewise, the activityof the plant ACCase has been demonstrated to be regulated byphosphorylation (Savage & Ohlrogge 1999, Plant J. 18:521-527) andalterations in the activity of the kinases and phosphatases (LMPs) thatact on the ACCase could lead to increased or decreased levels of seedlipid accumulation. Moreover, the presence of lipid kinase activities inchloroplast envelope membranes suggests that signal transductionpathways and/or membrane protein regulation occur in envelopes (see,e.g., Müller et al. 2000, J. Biol. Chem. 275:19475-19481 and literaturecited therein). The ABI1 and ABI2 genes encode two proteinserine/threonine phosphatases 2C, which are regulators in abscisic acidsignaling pathway, and thereby in early and late seed development (e.g.Merlot et al. 2001, Plant J. 25:295-303). For more examples see also thesection “Background of the Invention.”

The present invention also provides antibodies that specifically bind toan LMP-polypeptide, or a portion thereof, as encoded by a nucleic aciddisclosed herein or as described herein.

Antibodies can be made by many well-known methods (see, e.g. Harlow andLane, “Antibodies: A Laboratory Manual,” Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1988). Briefly, purified antigen can beinjected into an animal in an amount and in intervals sufficient toelicit an immune response. Antibodies can either be purified directly,or spleen cells can be obtained from the animal. The cells can thenfused with an immortal cell line and screened for antibody secretion.The antibodies can be used to screen nucleic acid clone libraries forcells secreting the antigen. Those positive clones can then be sequenced(see, for example, Kelly et al. 1992, Bio/Technology 10:163-167;Bebbington et al. 1992, Bio/Technology 10:169-175).

The phrase “selectively binds” with the polypeptide refers to a bindingreaction, which is determinative of the presence of the protein in aheterogeneous population of proteins and other biologics. Thus, underdesignated immunoassay conditions, the specified antibodies bound to aparticular protein do not bind in a significant amount to other proteinspresent in the sample. Selective binding to an antibody under suchconditions may require an antibody that is selected for its specificityfor a particular protein. A variety of immunoassay formats may be usedto select antibodies that selectively bind with a particular protein.For example, solid-phase ELISA immuno-assays are routinely used toselect antibodies selectively immunoreactive with a protein. See Harlowand Lane “Antibodies, A Laboratory Manual,” Cold Spring HarborPublications, New York (1988), for a description of immunoassay formatsand conditions that could be used to determine selective binding.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious hosts. A description of techniques for preparing such monoclonalantibodies may be found in Stites et al., editors, “Basic and ClinicalImmunology,” (Lange Medical Publications, Los Altos, Calif., FourthEdition) and references cited therein, and in Harlow and Lane(“Antibodies, A Laboratory Manual,” Cold Spring Harbor Publications, NewYork, 1988).

Throughout this application, various publications are referenced. Thedisclosures of all of these publications and those references citedwithin those publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art, to which this invention pertains.

FIGURES

FIGS. 1A-C: Nucleic acid sequence (SEQ ID NO: 1), open reading frame ofthe nucleic acid sequence (positions 1-2568 of SEQ ID NO: 1), and aminoacid sequence (SEQ ID NO: 2) of pk321AT01.

FIGS. 2A-C: Nucleic acid sequence (SEQ ID NO: 63), open reading frame ofthe nucleic acid sequence (positions 1-1218 of SEQ ID NO: 63), and aminoacid sequence (SEQ ID NO: 64) of pk322AT01.

FIGS. 3A-C: Nucleic acid sequence (SEQ ID NO: 87), open reading frame ofthe nucleic acid sequence (positions 1-927 of SEQ ID NO: 87), and aminoacid sequence (SEQ ID NO: 88) of pk323AT01.

FIGS. 4A-C: Nucleic acid sequence (SEQ ID NO: 158), open reading frameof the nucleic acid sequence (positions 1-1209 of SEQ ID NO: 158), andamino acid sequence (SEQ ID NO: 159) of pk324AT01.

FIGS. 5A-C: Nucleic acid sequence (SEQ ID NO: 238), open reading frameof the nucleic acid sequence (positions 1-1254 of SEQ ID NO: 238), andamino acid sequence (SEQ ID NO: 239) of pk325AT01.

FIGS. 6A-C: Nucleic acid sequence (SEQ ID NO: 273), open reading frameof the nucleic acid sequence (positions 1-1203 of SEQ ID NO: 273), andamino acid sequence (SEQ ID NO: 274) of pk326AT01.

FIGS. 7A-C: Nucleic acid sequence (SEQ ID NO: 321), open reading frameof the nucleic acid sequence (positions 1-798 of SEQ ID NO: 321), andamino acid sequence (SEQ ID NO: 322) of pk327AT01.

FIGS. 8A-C: Nucleic acid sequence (SEQ ID NO: 552), open reading frameof the nucleic acid sequence (positions 1-870 of SEQ ID NO: 552), andamino acid sequence (SEQ ID NO: 553) of pk328AT01.

FIGS. 9A-C: Nucleic acid sequence (SEQ ID NO: 591), open reading frameof the nucleic acid sequence (positions 1-753 of SEQ ID NO: 591), andamino acid sequence (SEQ ID NO: 592) of pk329AT01.

FIGS. 10A-C: Nucleic acid sequence (SEQ ID NO: 625), open reading frameof the nucleic acid sequence (positions 1-1608 of SEQ ID NO: 625), andamino acid sequence (SEQ ID NO: 626) of pk331AT01.

FIGS. 11A-C: Nucleic acid sequence (SEQ ID NO: 637), open reading frameof the nucleic acid sequence (positions 44-1633 of SEQ ID NO: 637), andamino acid sequence (SEQ ID NO: 638) of pk331GM59746258.

FIGS. 12A-C: Nucleic acid sequence (SEQ ID NO: 629), open reading frameof the nucleic acid sequence (positions 1-1557 of SEQ ID NO: 629), andamino acid sequence (SEQ ID NO: 630) of pk331OS37109650.

FIGS. 13A-C: Nucleic acid sequence (SEQ ID NO: 657), open reading frameof the nucleic acid sequence (positions 31-1509 of SEQ ID NO: 657), andamino acid sequence (SEQ ID NO: 658) of pk320AT01.

FIGS. 14A-C: Nucleic acid sequence (SEQ ID NO: 681), open reading frameof the nucleic acid sequence (positions 272-1765 of SEQ ID NO: 681), andamino acid sequence (SEQ ID NO: 682) of pk320BN51431102.

FIGS. 15A-C: Nucleic acid sequence (SEQ ID NO: 659), open reading frameof the nucleic acid sequence (positions 243-1742 of SEQ ID NO: 659), andamino acid sequence (SEQ ID NO: 660) of pk320GM59682267.

FIGS. 16A-C: Nucleic acid sequence (SEQ ID NO: 778), open reading frameof the nucleic acid sequence (positions 100-1812 of SEQ ID NO: 778), andamino acid sequence (SEQ ID NO: 779) of pk316BN44215842.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andExamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the claims included herein.

EXAMPLES Example 1 General Processes

a) General Cloning Processes

Cloning processes such as, for example, restriction cleavages, agarosegel electrophoresis, purification of DNA fragments, transfer of nucleicacids to nitrocellulose and nylon membranes, linkage of DNA fragments,transformation of Escherichia coli and yeast cells, growth of bacteriaand sequence analysis of recombinant DNA were carried out as describedin Sambrook et al. (1989, Cold Spring Harbor Laboratory Press: ISBN0-87969-309-6) or Kaiser, Michaelis and Mitchell (1994, “Methods inYeast Genetics,” Cold Spring Harbor Laboratory Press: ISBN0-87969-451-3).

b) Chemicals

The chemicals used were obtained, if not mentioned otherwise in thetext, in p.a. quality from the companies Fluka (Neu-Ulm), Merck(Darmstadt), Roth (Karlsruhe), Serva (Heidelberg) and Sigma(Deisenhofen). Solutions were prepared using purified, pyrogen-freewater, designated as H₂O in the following text, from a Milli-Q watersystem water purification plant (Millipore, Eschborn). Restrictionendonucleases, DNA-modifying enzymes, and molecular biology kits wereobtained from the companies AGS (Heidelberg), Amersham (Braunschweig),Biometra (Göttingen), Boehringer (Mannheim), Genomed (Bad Oeynnhausen),New England Biolabs (Schwalbach/Taunus), Novagen (Madison, Wis., USA),Perkin-Elmer (Weiterstadt), Pharmacia (Freiburg), Qiagen (Hilden), andStratagene (Amsterdam, Netherlands). They were used, if not mentionedotherwise, according to the manufacturer's instructions.

c) Plant Material and Growth:

Arabidopsis Plants

Arabidopsis thaliana cv Columbia were grown on plates with half-strengthMS medium (Murashige & Skoog, 1962, Physiol. Plant. 15:473-497), pH 6.2,2% sucrose and 0.8% agar. Seeds were sterilized for 20 minutes in 20%bleach 0.5% triton X100 and rinsed 6 times with excess sterile water.Wild type Arabidopsis seeds were preincubated for three days in the darkat 4° C. before placing them into an incubator (AR-75, PercivalScientific, Boone, Iowa) at a photon flux density of 60-80 μmol m⁻² s⁻¹and a light period of 16 hours (22° C.), and a dark period of 8 hours(18° C.). Plants were either grown as described above or on soil understandard conditions as described in Focks & Benning (1998, PlantPhysiol. 118:91-101).

Brassica napus

Brassica napus varieties AC Excel and Cresor were used for this study tocreate cDNA libraries. Seed, seed pod, flower, leaf, stem and roottissues were collected from plants that were in some cases dark-, salt-,heat- and drought-treated. However, this study focused on the use ofseed and seed-pod tissues for cDNA libraries. Plants were tagged toharvest seeds collected 60-75 days after planting from two time points:1-15 days and 15-25 days after anthesis. Plants have been grown inMetromix (Scotts, Marysville, Ohio) at 71° F. under a 14 hr photoperiod.Six seed and seed pod tissues of interest in this study were collectedto create the following cDNA libraries: Immature seeds, mature seeds,immature seed pods, mature seed pods, night-harvested seed pods andCresor variety (high erucic acid) seeds. Tissue samples were collectedwithin specified time points for each developing tissue and multiplesamples within a time frame pooled together for eventual extraction oftotal RNA. Samples from immature seeds were taken between 1-25 daysafter anthesis (daa), mature seeds between 25-50 daa, immature seed podsbetween 1-15 daa, mature seed pods between 15-50 daa, night-harvestedseed pods between 1-50 daa and Cresor seeds 5-25 daa.

Glycine max

Glycine max cv. Resnick was used for this study to create cDNAlibraries. Seed, seed pod, flower, leaf, stem and root tissues werecollected from plants that were in some cases dark-, salt-, heat- anddrought-treated. In some cases plants have been nematode infected aswell. However, this study focused on the use of seed and seed-podtissues for cDNA libraries. Plants were tagged to harvest seeds at theset days after anthesis: 5-15, 15-25, 25-35, & 33-50.

Oryza sativa

Oryza sativa ssp. Japonica cv. Nippon-barre was used for this study tocreate cDNA libraries. Seed, seed pod, flower, leaf, stem and roottissues were collected from plants that were in some cases dark-, salt-,heat- and drought-treated. This study focused on the use of seed embryotissues for cDNA libraries. Embryo and endosperm were collectedseparately in case endosperm tissue might interfere with RNA extraction.Plants have been grown in the greenhouse on Wisconsin soil (has highorganic matter) at 85° F. under a 14-h photoperiod. Rice embryos weredissected out of the developing seeds.

Example 2 Total DNA Isolation from Plants

The details for the isolation of total DNA relate to the working up ofone gram fresh weight of plant material.

CTAB buffer: 2% (w/v) N-cethyl-N,N,N-trimethylammonium bromide (CTAB);100 mM Tris HCl pH 8.0; 1.4 M NaCl; 20 mM EDTA. N-Laurylsarcosinebuffer: 10% (w/v) N-laurylsarcosine; 100 mM Tris HCl pH 8.0; 20 mM EDTA.

The plant material was triturated under liquid nitrogen in a mortar togive a fine powder and transferred to 2 ml Eppendorf vessels. The frozenplant material was then covered with a layer of 1 ml of decompositionbuffer (1 ml CTAB buffer, 100 μl of N-laurylsarcosine buffer, 20 μl ofβ-mercaptoethanol and 10 μl of proteinase K solution, 10 mg/ml) andincubated at 60° C. for one hour with continuous shaking. The homogenateobtained was distributed into two Eppendorf vessels (2 ml) and extractedtwice by shaking with the same volume of chloroform/isoamyl alcohol(24:1). For phase separation, centrifugation was carried out at 8000 gand RT for 15 min in each case. The DNA was then precipitated at −70° C.for 30 min using ice-cold isopropanol. The precipitated DNA wassedimented at 4° C. and 10,000 g for 30 min and resuspended in 180 μl ofTE buffer (Sambrook et al. 1989, Cold Spring Harbor Laboratory Press:ISBN 0-87969-309-6). For further purification, the DNA was treated withNaCl (1.2 M final concentration) and precipitated again at −70° C. for30 min using twice the volume of absolute ethanol. After a washing stepwith 70% ethanol, the DNA was dried and subsequently taken up in 50 μlof H₂O+RNAse (50 mg/ml final concentration). The DNA was dissolvedovernight at 4° C. and the RNAse digestion was subsequently carried outat 37° C. for 1 h. Storage of the DNA took place at 4° C.

Example 3 Isolation of Total RNA and poly-(A)+ RNA fromPlants—Arabidopsis thaliana

For the investigation of transcripts, both total RNA and poly-(A)+ RNAwere isolated. RNA is isolated from siliques of Arabidopsis plantsaccording to the following procedure:

RNA Preparation from Arabidopsis Seeds—“Hot” Extraction:

1. Buffers, enzymes and solution

-   -   2M KCl    -   Proteinase K        -   Phenol (for RNA)        -   Chloroform:Isoamylalcohol        -   (Phenol:choloroform 1:1; pH adjusted for RNA)        -   4 M LiCl, DEPC-treated        -   DEPC-treated water        -   3M NaOAc, pH 5, DEPC-treated        -   Isopropanol        -   70% ethanol (made up with DEPC-treated water)    -   Resuspension buffer: 0.5% SDS, 10 mM Tris pH 7.5, 1 mM EDTA made        up with DEPC-treated water as this solution can not be        DEPC-treated        -   Extraction Buffer:        -   0.2M Na Borate        -   30 mM EDTA        -   30 mM EGTA        -   1% SDS (250 μl of 10% SDS-solution for 2.5 ml buffer)        -   1% Deoxycholate (25 mg for 2.5 ml buffer)        -   2% PVPP (insoluble −50 mg for 2.5 ml buffer)        -   2% PVP 40K (50 mg for 2.5 ml buffer)        -   10 mM DTT            100 mM β-Mercaptoethanol (fresh, handle under fume hood—use            35 μl of 14.3M solution for 5 ml buffer)            2. Extraction. Heat extraction buffer up to 80° C. Grind            tissue in liquid nitrogen-cooled mortar, transfer tissue            powder to 1.5 ml tube. Tissue should kept frozen until            buffer is added so transfer the sample with pre-cooled            spatula and keep the tube in liquid nitrogen all time. Add            350 μl preheated extraction buffer (here for 100 mg tissue,            buffer volume can be as much as 500 μl for bigger samples)            to tube, vortex and heat tube to 80° C. for ˜1 min. Keep            then on ice. Vortex sample, grind additionally with electric            mortar.            3. Digestion. Add Proteinase K (0.15 mg/100 mg tissue),            vortex and keep at 37° C. for one hour.

First Purification. Add 27 μl 2M KCl. Chill on ice for 10 min.Centrifuge at 12.000 rpm for 10 minutes at room temperature. Transfersupernatant to fresh, RNAase-free tube and do one phenol extraction,followed by a chloroform:isoamylalcohol extraction. Add 1 vol.isopropanol to supernatant and chill on ice for 10 min. Pellet RNA bycentrifugation (7000 rpm for 10 min at RT). Resolve pellet in 1 ml 4MLiCl by 10 to 15 min vortexing. Pellet RNA by 5 min centrifugation.

Second Purification. Resuspend pellet in 500 μl Resuspension buffer. Add500 μl phenol and vortex. Add 250 μl chloroform:isoamylalcohol andvortex. Spin for 5 min. and transfer supernatant to fresh tube. Repeatchloform:isoamylalcohol extraction until interface is clear. Transfersupernatant to fresh tube and add 1/10 vol 3M NaOAc, pH 5, and 600 μlisopropanol. Keep at −20 for 20 min or longer. Pellet RNA by 10 mincentrifugation. Wash pellet once with 70% ethanol. Remove all remainingalcohol before resolving pellet with 15 to 20 μl DEPC-water. Determinequantity and quality by measuring the absorbance of a 1:200 dilution at260 and 280 nm. 40 μg RNA/ml=1OD260

RNA from wild-type of Arabidopsis is isolated as described (Hosein,2001, Plant Mol. Biol. Rep., 19:65a-65e; Ruuska, S. A., Girke, T.,Benning, C., & Ohlrogge, J. B., 2002, Plant Cell, 14:1191-1206).

The mRNA is prepared from total RNA, using the Amersham PharmaciaBiotech mRNA purification kit, which utilizes oligo(dT)-cellulosecolumns.

Isolation of Poly-(A)+ RNA was isolated using Dyna BeadsR (Dynal, Oslo,Norway) following the instructions of the manufacturer's protocol. Afterdetermination of the concentration of the RNA or of the poly(A)+ RNA,the RNA was precipitated by addition of 1/10 volumes of 3 M sodiumacetate pH 4.6 and 2 volumes of ethanol and stored at −70° C.

Brassica napus, Glycine max and Oryza sativa. Brassica napus and Glycinemax seeds were separated from pods to create homogeneous materials forseed and seed pod cDNA libraries. Tissues were ground into fine powderunder liquid N₂ using a mortar and pestle and transferred to a 50 mltube. Tissue samples were stored at −80° C. until extractions could beperformed. In the case of Oryza sativa, 5K-10K embryos and endospermwere isolated through dissection. Tissues were placed in small tubes orpetri dishes on ice during dissection. Containers were placed on dryice, then stored at −80° C.

Total RNA was extracted from tissues using RNeasy Maxi kit (Qiagen)according to manufacture's protocol and mRNA was processed from totalRNA using Oligotex mRNA Purification System kit (Qiagen), also accordingto manufacture's protocol. mRNA was sent to Hyseq PharmaceuticalsIncorporated (Sunnyville, Calif.) for further processing of mRNA fromeach tissue type into cDNA libraries and for use in their proprietaryprocesses, in which similar inserts in plasmids are clustered based onhybridization patterns.

Example 4 cDNA Library Construction

For cDNA library construction, first strand synthesis was achieved usingMurine Leukemia Virus reverse transcriptase (Roche, Mannheim, Germany)and oligo-d(T)-primers, second strand synthesis by incubation with DNApolymerase 1, Klenow enzyme and RNAseH digestion at 12° C. (2 h), 16° C.(1 h) and 22° C. (1 h). The reaction was stopped by incubation at 65° C.(10 min) and subsequently transferred to ice. Double stranded DNAmolecules were blunted by T4-DNA-polymerase (Roche, Mannheim) at 37° C.(30 min). Nucleotides were removed by phenol/chloroform extraction andSephadex G50 spin columns. EcoRI adapters (Pharmacia, Freiburg, Germany)were ligated to the cDNA ends by T4-DNA-ligase (Roche, 12° C.,overnight) and phosphorylated by incubation with polynucleotide kinase(Roche, 37° C., 30 min). This mixture was subjected to separation on alow melting agarose gel. DNA molecules larger than 300 base pairs wereeluted from the gel, phenol extracted, concentrated on Elutip-D-columns(Schleicher and Schuell, Dassel, Germany) and were ligated to vectorarms and packed into lambda ZAPII phages or lambda ZAP-Express phagesusing the Gigapack Gold Kit (Stratagene, Amsterdam, Netherlands) usingmaterial and following the instructions of the manufacturer.

Arabidopsis thaliana, Brassica napus, Glycine max and Oryza sativa cDNAlibraries were generated at Hyseq Pharmaceuticals Incorporated(Sunnyville, Calif.). No amplification steps were used in the libraryproduction to retain expression information. Hyseq's genomic approachinvolves grouping the genes into clusters and then sequencingrepresentative members from each cluster. cDNA libraries were generatedfrom oligo dT column purified mRNA. Colonies from transformation of thecDNA library into E. coli were randomly picked and the cDNA insert wereamplified by PCR and spotted on nylon membranes. A set of ³³-Pradiolabeled oligonucleotides was hybridized to the clones, and theresulting hybridization pattern determined, to which cluster aparticular clone belonged. cDNA clones and their DNA sequences wereobtained for use in overexpression in transgenic plants and in othermolecular biology processes described herein.

Example 5 Identification of LMP Genes of Interest of Arabidopsisthaliana, Brassica napus, Glycine max and Oryza sativa

This example illustrates how cDNA clones encoding LMP polypeptides ofArabidopsis thaliana, Brassica napus, Glycine max and Oryza sativa wereidentified and isolated. In order to identify Arabidopsis thaliana,Brassica napus, Glycine max and Oryza sativa LMP genes in proprietydatabases, a similarity analysis using BLAST software (Basic LocalAlignment Search Tool, version 2.2.6, Altschul et al., 1997, NucleicAcid Res. 25: 3389-3402)) was carried out. The default settings wereused except for e-value cut-off (1e-10) and all protein searches weredone using the BLOSUM62 matrix. The amino acid sequence of theArabidopsis LMP was used as a query to search and align DNA databasesfrom Arabidopsis thaliana, Brassica napus, Glycine max and Oryza sativathat were translated in all six reading frames, using the TBLASTNalgorithm. Such similarity analysis of BPS in-house databases resultedin the identification of numerous ESTs and cDNA contigs.

RNA expression profile data obtained from the Hyseq clustering processwere used to determine organ-specificity. Clones showing a greaterexpression in seed libraries compared to the other tissue libraries wereselected as LMP candidate genes. The Arabidopsis thaliana, Brassicanapus, Glycine max and Oryza sativa clones were selected foroverexpression in Arabidopsis.

Example 6 Cloning of Full-Length cDNAs and Orthologs of Identified LMPGenes

Clones corresponding to full-length sequences and partial cDNAs fromArabidopsis thaliana that were identified in Hyseq databases areisolated by RACE PCR using the SMART RACE cDNA amplification kit fromClontech allowing both 5′- and 3′ rapid amplification of cDNA ends(RACE). The isolation of cDNAs and the RACE PCR protocol used are basedon the manufacturer's conditions. The RACE product fragments areextracted from agarose gels with a QIAquick Gel Extraction Kit (Qiagen)and ligated into the TOPO pCR 2.1 vector (Invitrogen) followingmanufacturer's instructions. Recombinant vectors are transformed intoTOP10 cells (Invitrogen) using standard conditions (Sambrook et al.1989). Transformed cells are grown overnight at 37° C. on LB agarcontaining 50 μg/ml kanamycin and spread with 40 μl of a 40 mg/ml stocksolution of X-gal in dimethylformamide for blue-white selection. Singlewhite colonies are selected and used to inoculate 3 ml of liquid LBcontaining 50 μg/ml kanamycin and grown overnight at 37° C. Plasmid DNAis extracted using the QIAprep Spin Miniprep Kit (Qiagen) followingmanufacturer's instructions. Subsequent analyses of clones andrestriction mapping are performed according to standard molecularbiology techniques (Sambrook et al. 1989).

Clones of Arabidopsis thaliana, Brassica naups, Glycine max and Oryzasativa genes obtained from Hyseq were sequenced at using a ABI 377 slabgel sequencer and BigDye Terminator Ready Reaction kits (PE Biosystems,Foster City, Calif.). Gene specific primers were designed using thesesequences and genes were amplified from the plasmid supplied from Hysequsing touch-down PCR. In some cases, primers were designed to add an“AACA” Kozak-like sequence just upstream of the gene start codon and twobases downstream were, in some cases, changed to GC to facilitateincreased gene expression levels (Chandrashekhar et al., 1997, PlantMolecular Biology 35:993-1001). PCR reaction cycles were: 94° C., 5 min;9 cycles of 94° C., 1 min, 65° C., 1 min, 72° C., 4 min, and in whichthe anneal temperature was lowered by 1° C. each cycle; 20 cycles of 94°C., 1 min, 55° C., 1 min, 72° C., 4 min; and the PCR cycle was endedwith 72° C., 10 min. Amplified PCR products were gel purified from 1%agarose gels using GenElute-EtBr spin columns (Sigma) and after standardenzymatic digestion, were ligated into the plant binary vector pBPS-GB1for transformation into Arabidopsis thaliana or other crops. The binaryvector was amplified by overnight growth in E. coli DH5 in LB media andappropriate antibiotic and plasmid was prepared for downstream stepsusing Qiagen MiniPrep DNA preparation kit. The insert was verifiedthroughout the various cloning steps by determining its size throughrestriction digest and inserts were sequenced in parallel to planttransformations to ensure the expected gene was used in Arabidopsisthaliana or other crop transformation.

Gene sequences can be used to identify homologous or heterologous genes(orthologs, the same LMP gene from another plant) from cDNA or genomiclibraries. This can be done by designing PCR primers to conservedsequence regions identified by multiple sequence alignments. Orthologsare often identified by designing degenerate primers to full-length orpartial sequences of genes of interest.

Homologous genes (e.g. full-length cDNA clones) can be isolated vianucleic acid hybridization using for example cDNA libraries: Dependingon the abundance of the gene of interest, 100,000 up to 1,000,000recombinant bacteriophages are plated and transferred to nylonmembranes. After denaturation with alkali, DNA is immobilized on themembrane by, e.g., UV cross linking. Hybridization is carried out athigh stringency conditions. Aqueous solution hybridization and washingis performed at an ionic strength of 1 M NaCl and a temperature of 68°C. Hybridization probes are generated by, e.g., radioactive (32P) nicktranscription labeling (High Prime, Roche, Mannheim, Germany). Signalsare detected by autoradiography.

Partially homologous or heterologous genes that are related but notidentical can be identified in a procedure analogous to theabove-described procedure using low stringency hybridization and washingconditions. For aqueous hybridization, the ionic strength is normallykept at 1 M NaCl while the temperature is progressively lowered from 68°C. to 42° C.

Isolation of gene sequences with homologies (or sequenceidentity/similarity) only in a distinct domain of (for example 10-20amino acids) can be carried out by using synthetic radio labeledoligonucleotide probes. Radio labeled oligonucleotides are prepared byphosphorylation of the 5′ end of two complementary oligonucleotides withT4 polynucleotide kinase. The complementary oligonucleotides areannealed and ligated to form concatemers. The double-strandedconcatemers are than radiolabeled by for example nick transcription.Hybridization is normally performed at low stringency conditions usinghigh oligonucleotide concentrations.

Oligonucleotide Hybridization Solution:

-   -   6×SSC    -   0.01 M sodium phosphate    -   1 mM EDTA (pH 8)    -   0.5% SDS    -   100 μg/ml denaturated salmon sperm DNA    -   0.1% nonfat dried milk

During hybridization, temperature is lowered stepwise to 5-10° C. belowthe estimated oligonucleotide Tm or down to room temperature followed bywashing steps and autoradiography. Washing is performed with lowstringency, such as 3 washing steps using 4×SSC. Further details aredescribed by Sambrook et al. (1989, “Molecular Cloning: A LaboratoryManual,” Cold Spring Harbor Laboratory Press) or Ausubel et al. (1994,“Current Protocols in Molecular Biology,” John Wiley & Sons).

Example 7 Identification of Genes of Interest by Screening ExpressionLibraries with Antibodies

c-DNA clones can be used to produce recombinant protein for example inE. coli (e.g. Qiagen QIAexpress pQE system). Recombinant proteins arethen normally affinity purified via Ni-NTA affinity chromatography(Qiagen). Recombinant proteins can be used to produce specificantibodies for example by using standard techniques for rabbitimmunization. Antibodies are affinity purified using a Ni-NTA columnsaturated with the recombinant antigen as described by Gu et al. (1994,BioTechniques 17:257-262). The antibody can then be used to screenexpression cDNA libraries to identify homologous or heterologous genesvia an immunological screening (Sambrook et al. 1989, “MolecularCloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press orAusubel et al. 1994, “Current Protocols in Molecular Biology,” JohnWiley & Sons).

Example 8 Northern-Hybridization

For RNA hybridization, 20 μg of total RNA or 1 μg of poly-(A)+ RNA isseparated by gel electrophoresis in 1.25% agarose gels usingformaldehyde as described in Amasino (1986, Anal. Biochem. 152:304),transferred by capillary attraction using 10×SSC to positively chargednylon membranes (Hybond N+, Amersham, Braunschweig), immobilized by UVlight and pre-hybridized for 3 hours at 68° C. using hybridizationbuffer (10% dextran sulfate w/v, 1 M NaCl, 1% SDS, 100 μg/ml of herringsperm DNA). The labeling of the DNA probe with the Highprime DNAlabeling kit (Roche, Mannheim, Germany) is carried out during thepre-hybridization using alpha-32P dCTP (Amersham, Braunschweig,Germany). Hybridization is carried out after addition of the labeled DNAprobe in the same buffer at 68° C. overnight. The washing steps arecarried out twice for 15 min using 2×SSC and twice for 30 min using1×SSC, 1% SDS at 68° C. The exposure of the sealed filters is carriedout at −70° C. for a period of 1 day to 14 days.

Example 9 DNA Sequencing and Computational Functional Analysis

cDNA libraries can be used for DNA sequencing according to standardmethods, in particular by the chain termination method using the ABIPRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit(Perkin-Elmer, Weiterstadt, Germany). Random sequencing can be carriedout subsequent to preparative plasmid recovery from cDNA libraries viain vivo mass excision, retransformation, and subsequent plating of DH10Bon agar plates (material and protocol details from Stratagene,Amsterdam, Netherlands). Plasmid DNA can be prepared from overnightgrown E. coli cultures grown in Luria-Broth medium containing ampicillin(see Sambrook et al. (1989, Cold Spring Harbor Laboratory Press: ISBN0-87969-309-6) on a Qiagene DNA preparation robot (Qiagen, Hilden)according to the manufacturer's protocols). Sequences can be processedand annotated using the software package EST-MAX commercially providedby Bio-Max (Munich, Germany). The program incorporates bioinformaticsmethods important for functional and structural characterization ofprotein sequences. For reference see http://pedant.mips.biochem.mpg.de.

The most important algorithms incorporated in EST-MAX are: FASTA: Verysensitive protein sequence database searches with estimates ofstatistical significance (Pearson W. R. 1990, Rapid and sensitivesequence comparison with FASTP and FASTA. Methods Enzymol. 183:63-98).BLAST: Very sensitive protein sequence database searches with estimatesof statistical significance (Altschul S. F., Gish W., Miller W., MyersE. W. and Lipman D. J. Basic local alignment search tool. J. Mol. Biol.215:403-410). PREDATOR: High-accuracy secondary structure predictionfrom single and multiple sequences. (Frishman & Argos 1997, 75% accuracyin protein secondary structure prediction. Proteins 27:329-335).CLUSTALW: Multiple sequence alignment (Thompson, J. D., Higgins, D. G.and Gibson, T. J. 1994, “CLUSTAL W: improving the sensitivity ofprogressive multiple sequence alignment through sequence weighting,positions-specific gap penalties and weight matrix choice,” NucleicAcids Res. 22:4673-4680). TMAP: Transmembrane region prediction frommultiply aligned sequences (Persson B. & Argos P. 1994, “Prediction oftransmembrane segments in proteins utilizing multiple sequencealignments,” J. Mol. Biol. 237:182-192). ALOM2:Transmembrane regionprediction from single sequences (Klein P., Kanehisa M., and DeLisi C.1984, “Prediction of protein function from sequence properties: Adiscriminant analysis of a database,” Biochim. Biophys. Acta787:221-226. Version 2 by Dr. K. Nakai). PROSEARCH: Detection of PROSITEprotein sequence patterns. Kolakowski L. F. Jr., Leunissen J. A. M. andSmith J. E. 1992, “ProSearch: fast searching of protein sequences withregular expression patterns related to protein structure and function,”Biotechniques 13:919-921). BLIMPS: Similarity searches against adatabase of ungapped blocks (Wallace & Henikoff 1992, PATMAT: Asearching and extraction program for sequence, pattern and block queriesand databases, CABIOS 8:249-254. Written by Bill Alford).

Example 10 Plasmids for Plant Transformation

For plant transformation binary vectors such as pBinAR can be used(Höfgen & Willmitzer 1990, Plant Sci. 66:221-230). Construction of thebinary vectors can be performed by ligation of the cDNA in sense orantisense orientation into the T-DNA. 5′ to the cDNA a plant promoteractivates transcription of the cDNA. A polyadenylation sequence islocated 3′ to the cDNA. Tissue-specific expression can be achieved byusing a tissue specific promoter. For example, seed-specific expressioncan be achieved by cloning the napin or LeB4 or USP promoter 5′ to thecDNA. Also any other seed specific promoter element can be used. Forconstitutive expression within the whole plant the CaMV 35S promoter canbe used. The expressed protein can be targeted to a cellular compartmentusing a signal peptide, for example for plastids, mitochondria, orendoplasmic reticulum (Kermode 1996, Crit. Rev. Plant Sci. 15:285-423).The signal peptide is cloned 5′ in frame to the cDNA to achievesubcellular localization of the fusion protein.

Further examples for plant binary vectors are the pBPS-GB1, pSUN2-GW orpBPS-GB047 vectors, into which the LMP gene candidates are cloned. Thesebinary vectors contain an antibiotic resistance gene driven under thecontrol of the AtAct2-I promoter and a USP seed-specific promoter or aconstitutive promoter in front of the candidate gene with the NOSpAterminator or the OCS terminator. Partial or full-length LMP cDNA arecloned into the multiple cloning site of the plant binary vector insense or antisense orientation behind the USP seed-specific or PtxApromoters. The recombinant vector containing the gene of interest istransformed into Top10 cells (Invitrogen) using standard conditions.Transformed cells are selected for on LB agar containing 50 μg/mlkanamycin grown overnight at 37° C. Plasmid DNA is extracted using theQIAprep Spin Miniprep Kit (Qiagen) following manufacturer'sinstructions. Analysis of subsequent clones and restriction mapping isperformed according to standard molecular biology techniques (Sambrooket al. 1989, Molecular Cloning, A Laboratory Manual. 2^(nd) Edition.Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y.).

Example 11 Agrobacterium Mediated Plant Transformation

Agrobacterium mediated plant transformation with the LMP nucleic acidsdescribed herein can be performed using standard transformation andregeneration techniques (Gelvin, Stanton B. & Schilperoort R. A, PlantMolecular Biology Manual, 2nd ed. Kluwer Academic Publ., Dordrecht 1995in Sect., Ringbuc Zentrale Signatur: BT11-P; Glick, Bernard R. andThompson, John E. Methods in Plant Molecular Biology and Biotechnology,S. 360, CRC Press, Boca Raton 1993). For example, Agrobacterium mediatedtransformation can be performed using the GV3 (pMP90) (Koncz & Schell,1986, Mol. Gen. Genet. 204:383-396) or LBA4404 (Clontech) Agrobacteriumtumefaciens strain.

Arabidopsis thaliana can be grown and transformed according to standardconditions (Bechtold 1993, Acad. Sci. Paris. 316:1194-1199; Bent et al.1994, Science 265:1856-1860). Additionally, rapeseed can be transformedwith the LMR nucleic acids of the present invention via cotyledon orhypocotyl transformation (Moloney et al. 1989, Plant Cell Report8:238-242; De Block et al. 1989, Plant Physiol. 91:694-701). Use ofantibiotic for Agrobacterium and plant selection depends on the binaryvector and the Agrobacterium strain used for transformation. Rapeseedselection is normally performed using a selectable plant marker.Additionally, Agrobacterium mediated gene transfer to flax can beperformed using, for example, a technique described by Mlynarova et al.(1994, Plant Cell Report 13:282-285).

The LMP genes from various plant species were cloned into a binaryvector and expressed either under a constitutive promoter like thesuperpromoter (Stanton B. Gelvin, U.S. Pat. No. 5,428,147 and U.S. Pat.No. 5,217,903) or seed-specific promoters like USP (unknown seedprotein) from Vicia faba (Baeumlein et al. 1991, Mol. Gen. Genetics225:459-67), or the legumin B4 promoter (LeB4; Baeumlein et al. 1992,Plant J. 2:233-239) as well as promoters conferring seed-specificexpression in monocot plants like maize, barley, wheat, rye, rice etc.were used.

Transformation of soybean can be performed using for example a techniquedescribed in EP 0424047, U.S. Pat. No. 5,322,783 (Pioneer Hi-BredInternational) or in EP 0397687, U.S. Pat. No. 5,376,543, or U.S. Pat.No. 5,169,770 (University Toledo), or by any of a number of othertransformation procedures known in the art. Soybean seeds are surfacesterilized with 70% ethanol for 4 minutes at room temperature withcontinuous shaking, followed by 20% (v/v) CLOROX supplemented with 0.05%(v/v) TWEEN for 20 minutes with continuous shaking. Then the seeds arerinsed 4 times with distilled water and placed on moistened sterilefilter paper in a Petri dish at room temperature for 6 to 39 hours. Theseed coats are peeled off, and cotyledons are detached from the embryoaxis. The embryo axis is examined to make sure that the meristematicregion is not damaged. The excised embryo axes are collected in ahalf-open sterile Petri dish and air-dried to a moisture content lessthan 20% (fresh weight) in a sealed Petri dish until further use.

The method of plant transformation is also applicable to Brassica napusand other crops. In particular, seeds of canola are surface sterilizedwith 70% ethanol for 4 minutes at room temperature with continuousshaking, followed by 20% (v/v) CLOROX supplemented with 0.05% (v/v)TWEEN for 20 minutes, at room temperature with continuous shaking. Then,the seeds are rinsed 4 times with distilled water and placed onmoistened sterile filter paper in a Petri dish at room temperature for18 hours. The seed coats are removed and the seeds are air driedovernight in a half-open sterile Petri dish. During this period, theseeds lose approximately 85% of their water content. The seeds are thenstored at room temperature in a sealed Petri dish until further use.Agrobacterium tumefaciens culture is prepared from a single colony in LBsolid medium plus appropriate antibiotics (e.g. 100 mg/l streptomycin,50 mg/l kanamycin) followed by growth of the single colony in liquid LBmedium to an optical density at 600 nm of 0.8. Then, the bacteriaculture is pelleted at 7000 rpm for 7 minutes at room temperature, andre-suspended in MS (Murashige & Skoog 1962, Physiol. Plant. 15:473-497)medium supplemented with 100 mM acetosyringone. Bacteria cultures areincubated in this pre-induction medium for 2 hours at room temperaturebefore use. The axis of soybean zygotic seed embryos at approximately44% moisture content are imbibed for 2 h at room temperature with thepre-induced Agrobacterium suspension culture. (The imbibition of dryembryos with a culture of Agrobacterium is also applicable to maizeembryo axes). The embryos are removed from the imbibition culture andare transferred to Petri dishes containing solid MS medium supplementedwith 2% sucrose and incubated for 2 days, in the dark at roomtemperature. Alternatively, the embryos are placed on top of moistened(liquid MS medium) sterile filter paper in a Petri dish and incubatedunder the same conditions described above. After this period, theembryos are transferred to either solid or liquid MS medium supplementedwith 500 mg/l carbenicillin or 300 mg/l cefotaxime to kill theagrobacteria. The liquid medium is used to moisten the sterile filterpaper. The embryos are incubated during 4 weeks at 25° C., under 440μmol photons m⁻² s⁻¹ and 12 hours photoperiod. Once the seedlings haveproduced roots, they are transferred to sterile metromix soil. Themedium of the in vitro plants is washed off before transferring theplants to soil. The plants are kept under a plastic cover for 1 week tofavor the acclimatization process. Then the plants are transferred to agrowth room where they are incubated at 25° C., under 440 μmol m⁻²s⁻¹light intensity and 12 h photoperiod for about 80 days.

Samples of the primary transgenic plants (T₍ ₎) are analyzed by PCR toconfirm the presence of T-DNA. These results are confirmed by Southernhybridization wherein DNA is electrophoresed on a 1% agarose gel andtransferred to a positively charged nylon membrane (Roche Diagnostics).The PCR DIG Probe Synthesis Kit (Roche Diagnostics) is used to prepare adigoxigenin-labeled probe by PCR as recommended by the manufacturer.

As an example for monocot transformation, the construction of ptxApromoter (PF 55368-2 US, Song H. et al., 2004, application not yetpublished) in combination with maize Ubiquitin intron and LMP nucleicacid molecules is described. The PtxA-LMP gene construct in pUC isdigested with PacI and XmaI. pBPSMM348 is digested with PacI and XmaI toisolate maize Ubiquitin intron (ZmUbi intron) followed byelectrophoresis and the QIAEX II Gel Extraction Kit (cat #20021). TheZmUbi intron is ligated into the PtxA-pk331AT01 or PtxA-pk331OS37109650nucleic acid molecule in pUC to generate pUC based PtxA-ZmUbi intron-PCTor PCT-like nucleic acid molecule construct followed by restrictionenzyme digestion with AfeI and PmeI. PtxA-ZmUbi intron LMP gene cassetteis cut out of a Seaplaque low melting temperature agarose gel(SeaPlaque®GTG® Agarose catalog No. 50110) after electrophoresis. Amonocotyledonous base vector containing a selectable marker cassette(Monocot base vector) is digested with PmeI. The LMP nucleic acidmolecule expression cassette containing ptxA promoter-ZmUbi intron isligated into the Monocot base vector to generate PtxA-ZmUbi intron-LMPnucleic acid molecule construct. Subsequently, the PtxA-ZmUbi intron-LMPnucleic acid molecule construct is transformed into a recombinantLBA4404 strain containing pSB1 (super vir plasmid) using electroporationfollowing a general protocol in the art. Agrobacterium-mediatedtransformation in maize is performed using immature embryo following aprotocol described in U.S. Pat. No. 5,591,616. An imidazolinoneherbicide selection is applied to obtain transgenic maize lines.

In general, a rice (or other monocot) LMP gene under a plant promoter,like super promoter, could be transformed into corn, or another cropplant, to generate effects of monocot LMP genes in other monocots, ordicot LMP genes in other dicots, or monocot genes in dicots, or viceversa. The plasmids containing these LMP coding sequences, 5′ of apromoter and 3′ of a terminator would be constructed in a manner similarto those described for construction of other plasmids herein.

Example 12 In Vivo Mutagenesis

In vivo mutagenesis of microorganisms can be performed by incorporationand passage of the plasmid (or other vector) DNA through E. coli orother microorganisms (e.g. Bacillus spp. or yeasts such as Saccharomycescerevisiae) that are impaired in their capabilities to maintain theintegrity of their genetic information. Typical mutator strains havemutations in the genes for the DNA repair system (e.g., mutHLS, mutD,mutT, etc.; for reference, see Rupp W. D. 1996, “DNA repair mechanisms,”in: Escherichia coli and Salmonella, p. 2277-2294, ASM: Washington.)Such strains are well known to those skilled in the art. The use of suchstrains is illustrated, for example, in Greener and Callahan 1994,Strategies 7:32-34. Transfer of mutated DNA molecules into plants ispreferably done after selection and testing in microorganisms.Transgenic plants are generated according to various examples within theexemplification of this document.

Example 13 Assessment of the mRNA Expression and Activity of aRecombinant Gene Product in the Transformed Organism

The activity of a recombinant gene product in the transformed hostorganism can be measured on the transcriptional or/and on thetranslational level. A useful method to ascertain the level oftranscription of the gene (an indicator of the amount of mRNA availablefor translation to the gene product) is to perform a Northern blot (forreference see, for example, Ausubel et al. 1988, Current Protocols inMolecular Biology, Wiley: New York), in which a primer designed to bindto the gene of interest is labeled with a detectable tag (usuallyradioactive or chemiluminescent), such that when the total RNA of aculture of the organism is extracted, run on gel, transferred to astable matrix and incubated with this probe, the binding and quantity ofbinding of the probe indicates the presence and also the quantity ofmRNA for this gene. This information at least partially demonstrates thedegree of transcription of the transformed gene. Total cellular RNA canbe prepared from plant cells, tissues or organs by several methods, allwell-known in the art, such as that described in Bormann et al. (1992,Mol. Microbiol. 6:317-326).

To assess the presence or relative quantity of protein translated fromthis mRNA, standard techniques, such as a Western blot, may be employed(see, for example, Ausubel et al. 1988, Current Protocols in MolecularBiology, Wiley: New York). In this process, total cellular proteins areextracted, separated by gel electrophoresis, transferred to a matrixsuch as nitrocellulose, and incubated with a probe, such as an antibody,which specifically binds to the desired protein. This probe is generallytagged with a chemiluminescent or calorimetric label, which may bereadily detected. The presence and quantity of label observed indicatesthe presence and quantity of the desired mutant protein present in thecell.

The activity of LMPs that bind to DNA can be measured by severalwell-established methods, such as DNA band-shift assays (also called gelretardation assays). The effect of such LMP on the expression of othermolecules can be measured using reporter gene assays (such as thatdescribed in Kolmar H. et al. 1995, EMBO J. 14:3895-3904 and referencescited therein). Reporter gene test systems are well known andestablished for applications in both prokaryotic and eukaryotic cells,using enzymes such as beta-galactosidase, green fluorescent protein, andseveral others.

The determination of activity of lipid metabolism membrane-transportproteins can be performed according to techniques such as thosedescribed in Gennis R. B. (1989 Pores, Channels and Transporters, inBiomembranes, Molecular Structure and Function, Springer: Heidelberg,pp. 85-137, 199-234 and 270-322).

Example 14 In Vitro Analysis of the Function of Arabidopsis thaliana,Brassica napus, Glycine max and Oryza sativa LMP Genes in TransgenicPlants

The determination of activities and kinetic parameters of enzymes iswell established in the art. Experiments to determine the activity ofany given altered enzyme must be tailored to the specific activity ofthe wild-type enzyme, which is well within the ability of one skilled inthe art. Overviews about enzymes in general, as well as specific detailsconcerning structure, kinetics, principles, methods, applications andexamples for the determination of many enzyme activities may be found,for example, in the following references: Dixon, M. & Webb, E. C. 1979,Enzymes. Longmans: London; Fersht, (1985) Enzyme Structure andMechanism. Freeman: New York; Walsh (1979) Enzymatic ReactionMechanisms. Freeman: San Francisco; Price, N.C., Stevens, L. (1982)Fundamentals of Enzymology. Oxford Univ. Press: Oxford; Boyer, P. D.,ed. (1983) The Enzymes, 3rd ed. Academic Press: New York; Bisswanger,H., (1994) Enzymkinetik, 2nd ed. VCH: Weinheim (ISBN 3527300325);Bergmeyer, H. U., Bergmeyer, J., Graβl, M., eds. (1983-1986) Methods ofEnzymatic Analysis, 3rd ed., vol. I-XII, Verlag Chemie: Weinheim; andUllmann's Encyclopedia of Industrial Chemistry (1987) vol. A9, Enzymes.VCH: Weinheim, p. 352-363. For a more specific example for purification,preparation of membrane fractions, kinetic analyses and assay of PCTactivities see, for example, Marechal E, et al. (1997, Physiol. Plant.100:65-77) and literature cited therein.

Example 15 Analysis of the Impact of Recombinant Proteins on theProduction of a Desired Seed Storage Compound

Arabidopsis was used to investigate the influence of LMP genes on seedstorage compound accumulation. Seeds from transformed Arabidopsisthaliana plants were analyzed by gas chromatography (GC) for total oilcontent and fatty acid profile. Total fatty acid content of seeds ofcontrol and transgenic plants were measured with bulked seeds (usually 5mg seed weight) of a single plant. Three different types of controlshave been used: Col-2 (Columbia-2, the Arabidopsis ecotypes LMP gene ofinterest have been transformed in), C-24 (an Arabidopsis ecotype foundto accumulate high amounts of total fatty acids in seeds, used as apositive control herein) and BPS empty (without LMP gene of interest)binary vector construct. The controls indicated in the tables below havebeen grown side by side with the transgenic lines. Differences in thetotal values of the controls between different batches are explained bydifferences in the growth conditions, which were found to be verysensitive to small variations in the plant cultivation. Because of theseed bulking all values obtained with T2 seeds are the result of amixture of homozygous (for the gene of interest), heterozygous eventsand wild type seeds, implying that these data underestimate thepotential LMP gene effect. The binary vector pBPS-GB01 containing a USPpromoter driving the gene of interest has been used.

TABLE 1 Determination of the T2 seed total fatty acid content oftransgenic lines of pk321AT01 (containing SEQ ID NO: 1). The gene wasexpressed by a seed-specific promoter. Shown are the means (±standarddeviation). Average mean values are shown ± standard deviation, numberof individual measurements per plant line: 8-10; Col-2 is theArabidopsis ecotype the LMP gene has been transformed in, empty vectorcontrol is Col-2 transformed with a binary vector without gene ofinterest, C-24 is a high-oil Arabidopsis ecotype used as anotherpositive control. Transgenic seeds of pk321AT01 show a significantincrease relative to the empty vector control seeds (p < 0.05 asobtained by Student's t-test). Genotype g total fatty acids/g seedweight C-24 wild-type seeds 0.305 ± 0.022 Col-2 wild-type seeds 0.256 ±0.043 Empty vector control seeds 0.256 ± 0.035 pk321AT01 transgenicseeds 0.321 ± 0.019

TABLE 2 Determination of the T2 seed total fatty acid content oftransgenic lines of pk322AT01 (containing SEQ ID NO: 63). The gene wasexpressed by a seed-specific promoter. Shown are the means (±standarddeviation). Average mean values are shown ± standard deviation, numberof individual measurements per plant line: 8-20; Col-2 is theArabidopsis ecotype the LMP gene has been transformed in, empty vectorcontrol is Col-2 transformed with a binary vector without gene ofinterest, C-24 is a high-oil Arabidopsis ecotype used as anotherpositive control. Transgenic seeds of pk322AT01 show a significantincrease relative to the empty vector control seeds (p < 0.05 asobtained by Student's t-test). Genotype g total fatty acids/g seedweight C-24 wild-type seeds 0.305 ± 0.022 Col-2 wild-type seeds 0.256 ±0.043 Empty vector control seeds 0.256 ± 0.035 pk322AT01 transgenicseeds 0.325 ± 0.025

TABLE 3 Determination of the T2 seed total fatty acid content oftransgenic lines of pk323AT01 (containing SEQ ID NO: 87). The gene wasexpressed by a seed-specific promoter. Shown are the means (±standarddeviation). Average mean values are shown ± standard deviation, numberof individual measurements per plant line: 8-20; Col-2 is theArabidopsis ecotype the LMP gene has been transformed in, empty vectorcontrol is Col-2 transformed with a binary vector without gene ofinterest, C-24 is a high-oil Arabidopsis ecotype used as anotherpositive control. Transgenic seeds of pk323AT01 show a significantincrease relative to the empty vector control seeds (p < 0.05 asobtained by Student's t-test). g total fatty acids/ Genotype g seedweight C-24 wild-type seeds 0.305 ± 0.022 Col-2 wild-type seeds 0.256 ±0.043 Empty vector control seeds 0.256 ± 0.035 pk323AT01 transgenicseeds 0.299 ± 0.025

TABLE 4 Determination of the T2 seed total fatty acid content oftransgenic lines of pk324AT01 (containing SEQ ID NO: 158). The gene wasexpressed by a seed-specific promoter. Shown are the means (±standarddeviation). Average mean values are shown ± standard deviation, numberof individual measurements per plant line: 6-8; Col-2 is the Arabidopsisecotype the LMP gene has been transformed in, empty vector control isCol-2 transformed with a binary vector without gene of interest. g totalfatty acids/ Genotype g seed weight Col-2 wild-type seeds 0.311 ± 0.021Empty vector control seeds 0.324 ± 0.019 pk324AT01 transgenic seeds0.334 ± 0.011

TABLE 5 Determination of the T2 seed total fatty acid content oftransgenic lines of pk325AT01 (containing SEQ ID NO: 238). The gene wasexpressed by a seed-specific promoter. Shown are the means (±standarddeviation). Average mean values are shown ± standard deviation, numberof individual measurements per plant line: 6-10; Col-2 is theArabidopsis ecotype the LMP gene has been transformed in, empty vectorcontrol is Col-2 transformed with a binary vector without gene ofinterest, C-24 is a high-oil Arabidopsis ecotype used as anotherpositive control. Transgenic seeds of pk325AT01 show a significantincrease relative to the empty vector control seeds (p < 0.05 asobtained by Student's t-test). g total fatty acids/ Genotype g seedweight C-24 wild-type seeds 0.318 ± 0.017 Col-2 wild-type seeds 0.299 ±0.013 Empty vector control seeds 0.304 ± 0.023 pk325AT01 transgenicseeds 0.329 ± 0.010

TABLE 6 Determination of the T2 seed total fatty acid content oftransgenic lines of pk326AT01 (containing SEQ ID NO: 273). The gene wasexpressed by a seed-specific promoter. Shown are the means (±standarddeviation). Average mean values are shown ± standard deviation, numberof individual measurements per plant line: 6-10; Col-2 is theArabidopsis ecotype the LMP gene has been transformed in, empty vectorcontrol is Col-2 transformed with a binary vector without gene ofinterest, C-24 is a high-oil Arabidopsis ecotype used as anotherpositive control. Transgenic seeds of pk326AT01 show a significantincrease relative to the empty vector control seeds (p < 0.05 asobtained by Student's t-test). g total fatty acids/ Genotype g seedweight C-24 wild-type seeds 0.318 ± 0.017 Col-2 wild-type seeds 0.299 ±0.013 Empty vector control seeds 0.304 ± 0.023 pk326AT01 transgenicseeds 0.345 ± 0.010

TABLE 7 Determination of the T2 seed total fatty acid content oftransgenic lines of pk328AT01 (containing SEQ ID NO: 552). The gene wasexpressed by a seed-specific promoter. Shown are the means (±standarddeviation). Average mean values are shown ± standard deviation, numberof individual measurements per plant line: 6-20; Col-2 is theArabidopsis ecotype the LMP gene has been transformed in, empty vectorcontrol is Col-2 transformed with a binary vector without gene ofinterest, C-24 is a high-oil Arabidopsis ecotype used as anotherpositive control. Transgenic seeds of pk328AT01 show a significantincrease relative to the empty vector control seeds (p < 0.05 asobtained by Student's t-test). g total fatty acids/ Genotype g seedweight C-24 wild-type seeds 0.306 ± 0.021 Col-2 wild-type seeds 0.279 ±0.032 Empty vector control seeds 0.257 ± 0.033 pk328AT01 transgenicseeds 0.312 ± 0.033

TABLE 8a Determination of the T2 and T3 seed total fatty acid content oftransgenic lines of pk329AT01 (containing SEQ ID NO: 591). The gene wasexpressed by a seed specific promoter. Shown are the means (±standarddeviation). Average mean values are shown ± standard deviation, numberof individual measurements per plant line: 6-20; Col-2 is theArabidopsis ecotype the LMP gene has been transformed in, empty vectorcontrol is Col-2 transformed with a binary vector without gene ofinterest, C-24 is a high-oil Arabidopsis ecotype used as anotherpositive control. Transgenic seeds of pk329AT01 show a significantincrease relative to the empty vector control seeds (p < 0.05 asobtained by Student's t-test). g total fatty acids/ Genotype g seedweight T2 data: C-24 wild-type seeds 0.306 ± 0.021 Col-2 wild-type seeds0.279 ± 0.032 Empty vector control seeds 0.257 ± 0.033 pk329AT01transgenic seeds 0.305 ± 0.028 T3 data: C-24 wild-type seeds 0.343 ±0.017 Col-2 wild-type seeds 0.328 ± 0.028 Empty vector control seeds0.314 ± 0.024 pk329AT01-0957 transgenic seeds 0.343 ± 0.016pk329AT01-1114 transgenic seeds 0.345 ± 0.020

TABLE 8b Determination of the T3 seed total fatty acid content oftransgenic lines of pk329AT01 (containing SEQ ID NO: 591). The gene wasexpressed by a constitutive promoter. Shown are the means (±standarddeviation). Average mean values are shown ± standard deviation, numberof individual measurements per plant line: 6-20; Col-2 is theArabidopsis ecotype the LMP gene has been transformed in, empty vectorcontrol is Col-2 transformed with a binary vector without gene ofinterest, C-24 is a high-oil Arabidopsis ecotype used as anotherpositive control. Transgenic seeds of pk329AT01 show a significantincrease relative to the empty vector control seeds (p < 0.05 asobtained by Student's t-test). g total fatty acids/ Genotype g seedweight C-24 wild-type seeds 0.299 ± 0.020 Col-2 wild-type seeds 0.257 ±0.038 Empty vector control seeds 0.280 ± 0.035 pk329AT01-1232 transgenicseeds 0.298 ± 0.045 pk329AT01-1258 transgenic seeds 0.325 ± 0.040

TABLE 9a Determination of the T2 seed total fatty acid content oftransgenic lines of pk331AT01 (containing SEQ ID NO: 625). The gene wasexpressed by a seed-specific promoter. Shown are the means (±standarddeviation). Average mean values are shown ± standard deviation, numberof individual measurements per plant line: 6-25; Col-2 is theArabidopsis ecotype the LMP gene has been transformed in, empty vectorcontrol is Col-2 transformed with a binary vector without gene ofinterest, C-24 is a high-oil Arabidopsis ecotype used as anotherpositive control. Transgenic seeds of pk331AT01 show a significantincrease relative to the empty vector control seeds (p < 0.05 asobtained by Student's t-test). g total fatty acids/ Genotype g seedweight C-24 wild-type seeds 0.306 ± 0.021 Col-2 wild-type seeds 0.279 ±0.032 Empty vector control seeds 0.257 ± 0.033 pk331AT01 transgenicseeds 0.312 ± 0.027

TABLE 9b Determination of the T3 seed total fatty acid content oftransgenic lines of pk331AT01 (containing SEQ ID NO: 625). The gene wasexpressed by a constitutive promoter. Shown are the means (±standarddeviation). Average mean values are shown ± standard deviation, numberof individual measurements per plant line: 6-25; Col-2 is theArabidopsis ecotype the LMP gene has been transformed in, empty vectorcontrol is Col-2 transformed with a binary vector without gene ofinterest, C-24 is a high-oil Arabidopsis ecotype used as anotherpositive control. g total fatty acids/ Genotype g seed weight C-24wild-type seeds 0.299 ± 0.020 Col-2 wild-type seeds 0.257 ± 0.038 Emptyvector control seeds 0.287 ± 0.054 pk331AT01-1193 transgenic seeds 0.300± 0.038

TABLE 10 Determination of the T2 seed total fatty acid content oftransgenic lines of pk320BN 51431102 (containing SEQ ID NO: 681). Thegene was expressed by a seed-specific promoter. Shown are the means(±standard deviation). Average mean values are shown ± standarddeviation, number of individual measurements per plant line: 8-20; Col-2is the Arabidopsis ecotype the LMP gene has been transformed in, emptyvector control is Col-2 transformed with a binary vector without gene ofinterest. Transgenic seeds of pk320BN51431102 show a significantincrease relative to the empty vector control seeds (p < 0.05 asobtained by Student's t-test). g total fatty acids/ Genotype g seedweight Col-2 wild-type seeds 0.331 ± 0.014 Empty vector control seeds0.321 ± 0.025 pk320BN 51431102 transgenic seeds 0.352 ± 0.013

TABLE 11 Determination of the T2 seed total fatty acid content oftransgenic lines of pk320GM59682267 (containing SEQ ID NO: 659). Thegene was expressed by a seed-specific promoter. Shown are the means(±standard deviation). Average mean values are shown ± standarddeviation, number of individual measurements per plant line: 5-8; emptyvector control is Col-2 transformed with a binary vector without gene ofinterest, C-24 is a high-oil Arabidopsis ecotype used as anotherpositive control. Transgenic seeds of pk320GM59682267 show a significantincrease relative to the empty vector control seeds (p < 0.05 asobtained by Student's t-test). g total fatty acids/ Genotype g seedweight C-24 wild-type seeds 0.303 ± 0.044 Empty vector control seeds0.293 ± 0.031 pk320GM59682267 transgenic seeds 0.345 ± 0.029

TABLE 12 Determination of the T2 seed total fatty acid content oftransgenic lines of pk316BN 44215842 (containing SEQ ID NO: 778). Thegene was expressed by a seed-specific promoter. Shown are the means(±standard deviation). Average mean values are shown ± standarddeviation, number of individual measurements per plant line: 5-8; Col-2is the Arabidopsis ecotype the LMP gene has been transformed in, C-24 isa high-oil Arabidopsis ecotype used as another positive control.Transgenic seeds of pk316BN44215842 show a significant increase relativeto the empty vector control seeds (p < 0.05 as obtained by Student'st-test). g total fatty acids/ Genotype g seed weight C-24 wild-typeseeds 0.339 ± 0.022 Col-2 wild-type seeds 0.316 ± 0.011 pk316BN 44215842transgenic seeds 0.340 ± 0.017

The effect of the genetic modification in plants on a desired seedstorage compound (such as a sugar, lipid or fatty acid) can be assessedby growing the modified plant under suitable conditions and analyzingthe seeds or any other plant organ for increased production of thedesired product (i.e., a lipid or a fatty acid). Such analysistechniques are well known to one skilled in the art, and includespectroscopy, thin layer chromatography, staining methods of variouskinds, enzymatic and microbiological methods, and analyticalchromatography such as high performance liquid chromatography (see, forexample, Ullman 1985, Encyclopedia of Industrial Chemistry, vol. A2, pp.89-90 and 443-613, VCH: Weinheim; Fallon, A. et al. 1987, Applicationsof HPLC in Biochemistry in: Laboratory Techniques in Biochemistry andMolecular Biology, vol. 17; Rehm et al., 1993 Product recovery andpurification, Biotechnology, vol. 3, Chapter III, pp. 469-714, VCH:Weinheim; Belter, P. A. et al., 1988 Bioseparations: downstreamprocessing for biotechnology, John Wiley & Sons; Kennedy J. F. & CabralJ. M. S. 1992, Recovery processes for biological materials, John Wileyand Sons; Shaeiwitz J. A. & Henry J. D. 1988, Biochemical separationsin: Ulmann's Encyclopedia of Industrial Chemistry, Separation andpurification techniques in biotechnology, vol. B3, Chapter 11, pp. 1-27,VCH: Weinheim; and Dechow F. J. 1989).

Besides the above-mentioned methods, plant lipids are extracted fromplant material as described by Cahoon et al. (1999, Proc. Natl. Acad.Sci. USA 96, 22:12935-12940) and Browse et al. (1986, Anal. Biochemistry442:141-145). Qualitative and quantitative lipid or fatty acid analysisis described in Christie, William W., Advances in Lipid Methodology.Ayr/Scotland: Oily Press.—(Oily Press Lipid Library; Christie, WilliamW., Gas Chromatography and Lipids. A Practical Guide—Ayr, Scotland: OilyPress, 1989 Repr. 1992.—IX, 307 S.—(Oily Press Lipid Library; and“Progress in Lipid Research, Oxford: Pergamon Press, 1 (1952)-16 (1977)Progress in the Chemistry of Fats and Other Lipids CODEN.

Unequivocal proof of the presence of fatty acid products can be obtainedby the analysis of transgenic plants following standard analyticalprocedures: GC, GC-MS or TLC as variously described by Christie andreferences therein (1997 in: Advances on Lipid Methodology 4th ed.:Christie, Oily Press, Dundee, pp. 119-169; 1998). Detailed methods aredescribed for leaves by Lemieux et al. (1990, Theor. Appl. Genet.80:234-240) and for seeds by Focks & Benning (1998, Plant Physiol.118:91-101).

Positional analysis of the fatty acid composition at the sn-1, sn-2 orsn-3 positions of the glycerol backbone is determined by lipasedigestion (see, e.g., Siebertz & Heinz 1977, Z. Naturforsch.32c:193-205, and Christie 1987, Lipid Analysis 2^(nd) Edition, PergamonPress, Exeter, ISBN 0-08-023791-6).

Total seed oil levels can be measured by any appropriate method.Quantitation of seed oil contents is often performed with conventionalmethods, such as near infrared analysis (NIR) or nuclear magneticresonance imaging (NMR). NIR spectroscopy has become a standard methodfor screening seed samples whenever the samples of interest have beenamenable to this technique. Samples studied include canola, soybean,maize, wheat, rice, and others. NIR analysis of single seeds can be used(see e.g. Velasco et al., “Estimation of seed weight, oil content andfatty acid composition in intact single seeds of rapeseed (Brassicanapus L.) by near-infrared reflectance spectroscopy,” Euphytica, Vol.106, 1999, pp. 79-85). NMR has also been used to analyze oil content inseeds (see e.g. Robertson & Morrison, “Analysis of oil content ofsunflower seed by wide-line NMR,” Journal of the American Oil ChemistsSociety, 1979, Vol. 56, 1979, pp. 961-964, which is herein incorporatedby reference in its entirety).

A typical way to gather information regarding the influence of increasedor decreased protein activities on lipid and sugar biosynthetic pathwaysis for example via analyzing the carbon fluxes by labeling studies withleaves or seeds using ¹⁴C-acetate or ¹⁴C-pyruvate (see, e.g. Focks &Benning 1998, Plant Physiol. 118:91-101; Eccleston & Ohlrogge 1998,Plant Cell 10:613-621). The distribution of carbon-14 into lipids andaqueous soluble components can be determined by liquid scintillationcounting after the respective separation (for example on TLC plates)including standards like ⁴C-sucrose and ¹⁴C-malate (Eccleston & Ohlrogge1998, Plant Cell 10:613-621).

Material to be analyzed can be disintegrated via sonification, glassmilling, liquid nitrogen, and grinding or via other applicable methods.The material has to be centrifuged after disintegration. The sediment isre-suspended in distilled water, heated for 10 minutes at 100° C.,cooled on ice and centrifuged again followed by extraction in 0.5 Msulfuric acid in methanol containing 2% dimethoxypropane for 1 hour at90° C. leading to hydrolyzed oil and lipid compounds resulting intransmethylated lipids. These fatty acid methyl esters are extracted inpetrolether and finally subjected to GC analysis using a capillarycolumn (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 m, 0.32 mm) at atemperature gradient between 170° C. and 240° C. for 20 minutes and 5min. at 240° C. The identity of resulting fatty acid methylesters isdefined by the use of standards available form commercial sources (i.e.,Sigma).

In case of fatty acids where standards are not available, moleculeidentity is shown via derivatization and subsequent GC-MS analysis. Forexample, the localization of triple bond fatty acids is shown via GC-MSafter derivatization via 4,4-Dimethoxy-oxazolin-Derivaten (Christie,Oily Press, Dundee, 1998).

A common standard method for analyzing sugars, especially starch, ispublished by Stitt M., Lilley R. Mc. C., Gerhardt R. and Heldt M. W.(1989, “Determination of metabolite levels in specific cells andsubcellular compartments of plant leaves,” Methods Enzymol. 174:518-552;for other methods see also Härtel et al. 1998, Plant Physiol. Biochem.36:407-417 and Focks & Benning 1998, Plant Physiol. 118:91-101). For theextraction of soluble sugars and starch, 50 seeds are homogenized in 500μl of 80% (v/v) ethanol in a 1.5-ml polypropylene test tube andincubated at 70° C. for 90 min. Following centrifugation at 16,000 g for5 min, the supernatant is transferred to a new test tube. The pellet isextracted twice with 500 μl of 80% ethanol. The solvent of the combinedsupernatants is evaporated at room temperature under a vacuum. Theresidue is dissolved in 50 μl of water, representing the solublecarbohydrate fraction. The pellet left from the ethanol extraction,which contains the insoluble carbohydrates including starch, ishomogenized in 200 μl of 0.2 N KOH, and the suspension is incubated at95° C. for 1 h to dissolve the starch. Following the addition of 35 μlof 1 N acetic acid and centrifugation for 5 min at 16,000 g, thesupernatant is used for starch quantification.

To quantify soluble sugars, 10 μl of the sugar extract is added to 990μl of reaction buffer containing 100 mM imidazole, pH 6.9, 5 mM MgCl₂, 2mM NADP, 1 mM ATP, and 2 units 2 ml⁻¹ of Glucose-6-P-dehydrogenase. Forenzymatic determination of glucose, fructose, and sucrose, 4.5 units ofhexokinase, 1 unit of phosphoglucoisomerase, and 2 μl of a saturatedfructosidase solution are added in succession. The production of NADPHis photometrically monitored at a wavelength of 340 nm. Similarly,starch is assayed in 30 μl of the insoluble carbohydrate fraction with akit from Boehringer Mannheim.

An example for analyzing the protein content in leaves and seeds can befound by Bradford M. M. (1976, “A rapid and sensitive method for thequantification of microgram quantities of protein using the principle ofprotein dye binding,” Anal. Biochem. 72:248-254). For quantification oftotal seed protein, 15-20 seeds are homogenized in 250 μl of acetone ina 1.5-ml polypropylene test tube. Following centrifugation at 16,000 g,the supernatant is discarded and the vacuum-dried pellet is resuspendedin 250 μl of extraction buffer containing 50 mM Tris-HCl, pH 8.0, 250 mMNaCl, 1 mM EDTA, and 1% (w/v) SDS. Following incubation for 2 h at 25°C., the homogenate is centrifuged at 16,000 g for 5 min and 200 ml ofthe supernatant will be used for protein measurements. In the assay,γ-globulin is used for calibration. For protein measurements, Lowry DCprotein assay (Bio-Rad) or Bradford-assay (Bio-Rad) is used.

Enzymatic assays of hexokinase and fructokinase are performedspectrophotometrically according to Renz et al. (1993, Planta190:156-165), of phosphogluco-isomerase, ATP-dependent6-phosphofructokinase, pyrophosphate-dependent 6-phospho-fructokinase,Fructose-1,6-bisphosphate aldolase, triose phosphate isomerase,glyceral-3-P dehydrogenase, phosphoglycerate kinase, phosphoglyceratemutase, enolase and pyruvate kinase are performed according to Burrellet al. (1994, Planta 194:95-101) and of UDP-Glucose-pyrophosphorylaseaccording to Zrenner et al. (1995, Plant J. 7:97-107).

Enzymatic activities of phosphatidate cytidylyltransferase can bedetermined as described (e.g. Sturton R G & Brindley D N, 1977, Biochem.J. 162:25-32; Kelley M J & Carman G M 1987, J. Biol. Chem.262:14563-14570).

Intermediates of the carbohydrate metabolism, like glucose-1-phosphate,glucose-6-phosphate, fructose-6-phosphate, phosphoenol-pyruvate,pyruvate, and ATP are measured as described in Härtel et al. (1998,Plant Physiol. Biochem. 36:407-417) and metabolites are measured asdescribed in Jelitto et al. (1992, Planta 188:238-244).

In addition to the measurement of the final seed storage compound (I.e.,lipid, starch or storage protein) it is also possible to analyze othercomponents of the metabolic pathways utilized for the production of adesired seed storage compound, such as intermediates and side-products,to determine the overall efficiency of production of the compound (Fiehnet al. 2000, Nature Biotech. 18:1447-1161).

For example, yeast expression vectors comprising the nucleic acidsdisclosed herein, or fragments thereof, can be constructed andtransformed into Saccharomyces cerevisiae using standard protocols. Theresulting transgenic cells can then be assayed for alterations in sugar,oil, lipid, or fatty acid contents.

Similarly, plant expression vectors comprising the nucleic acidsdisclosed herein, or fragments thereof, can be constructed andtransformed into an appropriate plant cell such as Arabidopsis, soybean,rapeseed, rice, linseed, maize, barley, wheat, Medicago truncatula,etc., using standard protocols. The resulting transgenic cells and/orplants derived there from can then be assayed for alterations in sugar,oil, lipid, or fatty acid contents.

Additionally, the sequences disclosed herein, or fragments thereof, canbe used to generate knockout mutations in the genomes of variousorganisms, such as bacteria, mammalian cells, yeast cells, and plantcells (Girke at al. 1998, Plant J. 15:39-48). The resultant knockoutcells can then be evaluated for their composition and content in seedstorage compounds, and the effect on the phenotype and/or genotype ofthe mutation. For other methods of gene inactivation include U.S. Pat.No. 6,004,804, “Non-Chimeric Mutational Vectors,” and Puttaraju et al.(1999, “Spliceosome-mediated RNA trans-splicing as a tool for genetherapy,” Nature Biotech. 17:246-252).

Example 16 Purification of the Desired Product from TransformedOrganisms

An LMP can be recovered from plant material by various methods wellknown in the art. Organs of plants can be separated mechanically fromother tissue or organs prior to isolation of the seed storage compoundfrom the plant organ. Following homogenization of the tissue, cellulardebris is removed by centrifugation and the supernatant fractioncontaining the soluble proteins is retained for further purification ofthe desired compound. If the product is secreted from cells grown inculture, then the cells are removed from the culture by low-speedcentrifugation and the supernate fraction is retained for furtherpurification.

The supernatant fraction from either purification method is subjected tochromatography with a suitable resin, in which the desired molecule iseither retained on a chromatography resin while many of the impuritiesin the sample are not, or where the impurities are retained by theresin, while the sample is not. Such chromatography steps may berepeated as necessary, using the same or different chromatographyresins. One skilled in the art would be well-versed in the selection ofappropriate chromatography resins and in their most efficaciousapplication for a particular molecule to be purified. The purifiedproduct may be concentrated by filtration or ultrafiltration, and storedat a temperature at which the stability of the product is maximized.

There is a wide array of purification methods known to the art and thepreceding method of purification is not meant to be limiting. Suchpurification techniques are described, for example, in Bailey J. E. &Ollis D. F. 1986, Biochemical Engineering Fundamentals, McGraw-Hill: NewYork).

The identity and purity of the isolated compounds may be assessed bytechniques standard in the art. These include high-performance liquidchromatography (HPLC), spectroscopic methods, staining methods, thinlayer chromatography, analytical chromatography such as high performanceliquid chromatography, NIRS, enzymatic assay, or microbiologically. Suchanalysis methods are reviewed in: Patek et al. (1994, Appl. Environ.Microbiol. 60:133-140), Malakhova et al. (1996, Biotekhnologiya11:27-32) and Schmidt et al. (1998, Bioprocess Engineer 19:67-70),Ulmann's Encyclopedia of Industrial Chemistry (1996, Vol. A27, VCH:Weinheim, p. 89-90, p. 521-540, p. 540-547, p. 559-566, 575-581 and p.581-587) and Michal G. (1999, Biochemical Pathways: An Atlas ofBiochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. etal. 1987, Applications of HPLC in Biochemistry in: Laboratory Techniquesin Biochemistry and Molecular Biology, vol. 17).

Example 17 Screening for Increased Stress Tolerance and Plant Growth

The transgenic plants are screened for their improved stress tolerancedemonstrating that transgene expression confers stress tolerance. Thetransgenic plants are further screened for their growth ratedemonstrating that transgene expression confers increased growth ratesand/or increased seed yield.

Increased seed size might be reflected in an increased seed weight ofgene overexpressors. Increased seed size leads to greater yield in manyeconomically important crop plants. Therefore, increased seed size isone goal of genetic engineering and selection using LMPs as described inthis application.

For in vitro root analysis square plates measuring 12 cm×12 cm can beused. For each plate, 52 ml of MS media (0.5×MS salts, 0.5% sucrose, 0.5g/L MES buffer, 1% Phytagar) without selection will be used. Plates willbe allowed to dry in the sterile hood for one hour to reduce futurecondensation.

Seed aliquots will be sterilized in glass vials with ethanol for 5minutes, the ethanol was removed, and the seeds were allowed to dry inthe sterile hood for one hour.

Seeds will be spotted in the plates using the Vacuseed Device (Lehle).After the seeds were spotted on the plates, the plates will be wrappedwith Ventwrap and placed vertically in racks in the dark at 4° C. forfour days to stratify the seeds. The plates are transferred to a C5Percival Growth Chamber and placed vertically. The growth chamberconditions will be 23° C. day/21° C. night and 16 h day/8 h night.

For data collection a high resolution flat-bed scanner is used. Analysisof the roots is done using the WinRhizo software package.

For soil root analysis seeds may be imbibed at 4° C. for 2 days in waterand planted directly in soil with no selection. Deepots (Hummert D40)will be used with a saturated peat pellet (Jiffy 727) at the base andfilled with water saturated Metromix. After planting, pots will becovered with plastic wrap to prevent drying. Plants may be grown usingonly water present at media preparation, as the water in the soil inthese large pots is sufficient for 3 weeks of growth, and encouragesrapid root growth. The plastic wrapping of the pots will be removedafter 12 days and morphological data documented. At day 17 the aerialparts of the plant will be harvested, dried (65° C. for 2 days) and dryweight measured. To examine the roots the peat pellet will be pushedtowards the top of the pot to remove the soil and roots as a unit. Thesoil will then be separated from the roots in a tray and the maximumroot length will be measured. Root length of all plants for alltransgenic lines will be averaged and compared against the average ofthe wild type plants.

TABLE 13 Plant Lipid Classes Neutral Lipids Triacylglycerol (TAG)Diacylglycerol (DAG) Monoacylglycerol (MAG) Polar LipidsMonogalactosyldiacylglycerol (MGDG) Digalactosyldiacylglycerol (DGDG)Phosphatidylglycerol (PG) Phosphatidylcholine (PC)Phosphatidylethanolamine (PE) Phosphatidylinositol (PI)Phosphatidylserine (PS) Sulfoquinovosyldiacylglycerol

TABLE 14 Common Plant Fatty Acids 16:0 Palmitic acid 16:1 Palmitoleicacid 16:3 Palmitolenic acid 18:0 Stearic acid 18:1 Oleic acid 18:2Linoleic acid 18:3 Linolenic acid γ-18:3    Gamma-linolenic acid* 20:0Arachidic acid 20:1 Eicosenoic acid 22:6 Docosahexanoic acid (DHA)* 20:2Eicosadienoic acid 20:4 Arachidonic acid (AA)* 20:5 Eicosapentaenoicacid (EPA)* 22:1 Erucic acid *These fatty acids do not normally occur inplant seed oils, but their production in transgenic plant seed oil is ofimportance in plant biotechnology.

TABLE 15 A table of the putative activities of the LMPs Seq Sequence ORFID name Species Function position 1 pk321AT 01 Arabidopsis AtTPS1 gene,trehalose-6-phosphate synthase 1-2568 thaliana 63 pk322AT 01 Arabidopsisputative pyruvate dehydrogenase E1 beta 1-1218 thaliana subunit 87pk323AT 01 Arabidopsis phosphate/phosphoenolpyruvate translocator 1-927thaliana like protein 158 pk324AT 01 Arabidopsis chloroplastNAD-dependent malate 1-1209 thaliana dehydrogenase 238 pk325AT 01Arabidopsis magnesium chelatase subunit of 1-1254 thalianaprotochlorophyllide reductase 273 pk326AT 01 Arabidopsisprotochlorophyllide reductase precursor 1-1203 thaliana 321 pk327AT 01Arabidopsis putative photosystem II type I chlorophyll a/b 1-798 thaliana binding protein 552 pk328AT 01 Arabidopsis chlorophylla/b-binding protein CP29 1-870  thaliana 591 pk329AT 01 ArabidopsisCHLOROPHYLL A-B BINDING PROTEIN 4 1-753  thaliana PRECURSOR homolog 625pk331AT 01 Arabidopsis Arabidopsis thaliana chlorophyll a oxygenase1-1608 thaliana (CAO)/chlorophyll b synthase 637 pk331GM Glycine maxChlorophyll a oxygenase (CAO)/chlorophyll b 44-1633  59746258 synthase629 pk331OS Oryza sativa Chlorophyll a oxygenase (CAO)/chlorophyll b82-1704  37109650 synthase 657 pk320AT 01 Arabidopsis Hexokinase-likeprotein 31-1509  thaliana 681 pk320BN Brassica Hexokinase-like protein123-1625  51431102 napus 659 pk320GM59682267 Glycine max Hexokinase-likeprotein 179-1666  778 pk316BN Brassica Pyruvate kinase 100-1812 44215842 napus

Those skilled in the art will recognize, or will be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the claims to the invention disclosedand claimed herein.

The invention claimed is:
 1. A method for the manufacture of apolypeptide capable of increasing the content of total fatty acids inseed when expressed in a transgenic plant relative to a correspondingcontrol plant comprising: (a) expressing in a host cell apolynucleotide, wherein the polynucleotide comprises a nucleic acidsequence selected from the group consisting of: (i) the nucleic acidsequence of SEQ ID NO: 778; (ii) a nucleic acid sequence encoding apolypeptide comprising the amino acid sequence of SEQ ID NO: 779; and(iii) a nucleic acid sequence which is at least 90% identical to thenucleic acid sequence of (i) or (ii), wherein said nucleic acid sequenceencodes a polypeptide capable of increasing the content of total fattyacids in seed when expressed in a transgenic plant relative to acorresponding control plant; and (b) obtaining the polypeptide encodedby said polynucleotide from the host cell.
 2. A method for themanufacture of a lipid or a fatty acid in a plant cell, plant, or partthereof, or progeny therefrom comprising: (a) expressing in a transgenicplant cell, plant, or part thereof, or progeny therefrom apolynucleotide, wherein the polynucleotide comprises a nucleic acidsequence selected from the group consisting of: (i) the nucleic acidsequence of SEQ ID NO: 778; (ii) a nucleic acid sequence encoding apolypeptide comprising the amino acid sequence of SEQ ID NO: 779; and(iii) a nucleic acid sequence which is at least 90% identical to thenucleic acid sequence of (i) or (ii), wherein said nucleic acid sequenceencodes a polypeptide capable of increasing the content of total fattyacids in seed when expressed in a transgenic plant; (b) cultivating thetransgenic plant cell, plant, or part thereof, or progeny therefrom of(a) under conditions allowing synthesis of said fatty acid; and (c)obtaining said fatty acid from the transgenic plant cell, plant, or partthereof, or progeny therefrom.
 3. A method for the manufacture of aplant having increased content of total fatty acids in seed of a plantrelative to a corresponding control plant comprising: (a) introducinginto a plant cell a polynucleotide, wherein the polynucleotide comprisesa nucleic acid sequence selected from the group consisting of: (i) thenucleic acid sequence of SEQ ID NO: 778; (ii) a nucleic acid sequenceencoding a polypeptide comprising the amino acid sequence of SEQ ID NO:779; and (iv) a nucleic acid sequence which is at least 90% identical tothe nucleic acid sequence of (i) or (ii), wherein said nucleic acidsequence encodes a polypeptide capable of increasing the content oftotal fatty acids in seed when expressed in a transgenic plant relativeto a corresponding control plant; and (b) generating a transgenic plantfrom said plant cell, wherein the polypeptide encoded by thepolynucleotide when expressed in the transgenic plant increases thecontent of total fatty acids in seed of the transgenic plant relative toa corresponding control plant.
 4. The method of claim 1, wherein thepolynucleotide comprises a nucleic acid sequence which is at least 95%identical to the nucleic acid sequence of (i) or (ii), wherein saidnucleic acid sequence encodes a polypeptide capable of increasing thecontent of total fatty acids in seed when expressed in a transgenicplant relative to a corresponding control plant.
 5. The method of claim2, wherein the polynucleotide comprises a nucleic acid sequence which isat least 95% identical to the nucleic acid sequence of (i) or (ii),wherein said nucleic acid sequence encodes a polypeptide capable ofincreasing the content of total fatty acids in seed when expressed in atransgenic plant relative to a corresponding control plant.
 6. Themethod of claim 3, wherein the polynucleotide comprises a nucleic acidsequence which is at least 95% identical to the nucleic acid sequence of(i) or (ii), wherein said nucleic acid sequence encodes a polypeptidecapable of increasing the content of total fatty acids in seed whenexpressed in a transgenic plant relative to a corresponding controlplant.
 7. The method of claim 1, wherein the polynucleotide comprisesthe nucleic acid sequence of (i) or (ii), wherein said nucleic acidsequence encodes a polypeptide capable of increasing the content oftotal fatty acids in seed when expressed in a transgenic plant relativeto a corresponding control plant.
 8. The method of claim 2, wherein thepolynucleotide comprises the nucleic acid sequence of (i) or (ii),wherein said nucleic acid sequence encodes a polypeptide capable ofincreasing the content of total fatty acids in seed when expressed in atransgenic plant relative to a corresponding control plant.
 9. Themethod of claim 3, wherein the polynucleotide comprises the nucleic acidsequence of (i) or (ii), wherein said nucleic acid sequence encodes apolypeptide capable of increasing the content of total fatty acids inseed when expressed in a transgenic plant relative to a correspondingcontrol plant.
 10. The method of claim 1, wherein the polynucleotidecomprises a nucleic acid sequence encoding a polypeptide comprising anamino acid sequence having greater than 95% sequence identity to theamino acid sequence of SEQ ID NO: 779, wherein said nucleic acidsequence encodes a polypeptide capable of increasing the content oftotal fatty acids in seed when expressed in a transgenic plant relativeto a corresponding control plant.
 11. The method of claim 2, wherein thepolynucleotide comprises a nucleic acid sequence encoding a polypeptidecomprising an amino acid sequence having greater than 95% sequenceidentity to the amino acid sequence of SEQ ID NO: 779, wherein saidnucleic acid sequence encodes a polypeptide capable of increasing thecontent of total fatty acids in seed when expressed in a transgenicplant relative to a corresponding control plant.
 12. The method of claim3, wherein the polynucleotide comprises a nucleic acid sequence encodinga polypeptide comprising an amino acid sequence having greater than 95%sequence identity to the amino acid sequence of SEQ ID NO: 779, whereinsaid nucleic acid sequence encodes a polypeptide capable of increasingthe content of total fatty acids in seed when expressed in a transgenicplant relative to a corresponding control plant.
 13. The method of claim3, further comprising obtaining transgenic progeny from the transgenicplant, or transgenic seed from the transgenic plant or from progenytherefrom, wherein the transgenic progeny or transgenic seed comprisethe polynucleotide.
 14. The method of claim 3, further comprisingselecting a transgenic plant or transgenic progeny therefrom havingincreased content of total fatty acids relative to a correspondingcontrol plant, wherein the transgenic plant or transgenic progenycomprise the polynucleotide.
 15. The method of claim 1, wherein thepolynucleotide is operably linked to a seed-specific promoter.
 16. Themethod of claim 2, wherein the polynucleotide is operably linked to aseed-specific promoter.
 17. The method of claim 3, wherein thepolynucleotide is operably linked to a seed-specific promoter.